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Histology Laboratory Manual 2016-2017: College of Physicians and Surgeons Columbia University
Histology Laboratory Manual 2016-2017: College of Physicians and Surgeons Columbia University
2016-2017
Patrice F Spitalnik MD
Histology Director
pfs2101@Columbia.edu
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LABORATORY TOPICS
1. INTRODUCTION: MICROSCOPY, CELLS, ORGANELLES, MITOSIS 5
2. EPITHELIUM 9
3. CONNECTIVE TISSUE 13
4. CARTILAGE, BONE, BONE DEVELOPMENT 17
5. NERVE 25
6. MUSCLE 30
7. BLOOD, HEMATOPOIESIS, BONE MARROW 35
8. LYMPHOID TISSUES 40
9. CARDIOVASCULAR SYSTEM 46
10. SKIN 51
11. RESPIRATORY SYSTEM 54
12. URINARY SYSTEM 57
13. ENDOCRINE GLANDS 61
14. MALE REPRODUCTIVE SYSTEM 66
15. FEMALE REPRODUCTIVE SYSTEM 69
16. GASTROINTESTINAL SYSTEM I: DIGESTIVE TRACT 76
17. GASTROINTESTINAL SYSTEM II: ASSOCIATED ORGANS 82
APPENDIX
ANSWERS TO QUESTIONS 86
HISTOLOGICAL TECHNIQUES 97
THE MICROSCOPE 104
HISTOLOGY GLASS SLIDE COLLECTION 106
Acknowledgements:
Caitlin Alexander edited the text and created many of the diagrams and micrographs as part of
the Columbia College of Physicians and Surgeons Scholarly Project Program 2015-2016.
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As the structural and functional relationships of various cells, tissues, and organs are
considered throughout the course, always be aware of one simple concept: All of the tissues
and organs of the body are composed of cells and the extracellular products of cells (the
matrix). It is for this reason that we emphasize the basic components of cells and their
matrices during the early portion of the course. With an understanding of the nature of the
relationship between cells and their matrices, we can proceed to the study of the organization
of these two components into the basic tissues of the body.
There are only four basic tissues in the body, although each category can be subdivided. In
turn, the four basic tissues are organized into the various organs of the body, and these
generally exist as interrelated functional units termed organ systems.
1) Epithelium
2) Connective tissue
3) Muscle
4) Nervous tissue
Again, we emphasize: All of the organs of the body are composed of varying proportions of the
four basic tissues, and each of the four basic tissues consists of cells and extracellular
matrices. This simple concept is fundamental to the study of histology.
MANUAL
This manual is a guide to work in the histology laboratory. For each topic there is a brief
introduction. This is followed by a list of images, with commentary.
Note: The images were scanned from the Histology Slide Collection, which is listed at the end
of this manual. Sets of these slides and microscopes are available for those who wish to use
them. A few slides were not scanned (indicated by an asterisk).
TEXTBOOK
In this course you will have access to an iBook that is available in Courseworks. This book
includes some images that are not in the online lab manual and supplements the basic
material.
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Learning objectives:
1. Understand and be able to describe how the most common dye combination, hematoxylin
and eosin (H&E), stains various components of cells and tissues.
2. Identify cells and subcellular organelles.
3. Relate the appearance of a cell as seen with the light microscope (LM) with that at with the
electron microscope (TEM, transmission electron microscope).
4. Recognize and understand the stages in mitosis.
CELLS, ORGANELLES
Slide Preparation:
The first step in preparing a tissue or organ for microscopic examination is fixation, or
preservation, of the specimen. Formalin is a commonly used fixative. Many other fixatives are
available and are used in the study of specific structures.
The specimen on the microscope slide is a thin section (usually 5 micrometers) of the fixed
tissue or organ. The section is stained by one or more dyes. Without staining the section
would be nearly invisible with the microscope. Components of the specimen generally stain
selectively and, on this basis, various regions of the specimen may be differentiated from each
other.
Most dyes are neutral salts. In some stains, the dye moiety is a cation and such dyes are
called cationic or basic dyes. These form salts with tissue anions, especially the phosphate
groups of the nucleic acids and the sulfate groups of the glycosaminoglycans. When the dye
moiety is an anion, the dye is called anionic or acid dye and salt formation occurs with tissue
cations including the lysine and arginine groups of tissue proteins. Tissue components that
recognize basic dyes are "basophilic" and those that recognize acid dyes are "acidophilic".
A common combination of stains is hematoxylin and eosin (H&E), which are commonly
referred to as basic and acid dyes, respectively.
HISTOCHEMISTRY
In the diagram below identify the base and apex of the cells of a secretory unit, the acinus or
gland. Note the basophilia in the basal compartment and the acidophilia in the apical (luminal)
compartment of the cytoplasm. What subcellular organelle is responsible for attracting the
basic stain?
Pancreatic Acinus
Examine the cells lining the lumen of this organ, the gall
bladder. The apical border of these cells faces the lumen. A
border may be identified at the apex of the cells, which has
slightly different optical properties from the remainder of the cell.
Under optimum conditions faint striations, oriented parallel to the
long axis of the cell, are seen in the border. These are difficult to
resolve at the light microscopic level, but with electron
microscopy, these striations are seen to be precisely arranged
microvilli, containing cores of actin filaments. Why are the
microvilli not visible on all cells lining the lumen?
40x view of
#117 Small intestine, H&E - Microvilli gallbladder mucosa
Study the cells lining the lumen of the small intestine as another example of a microvillous
border. The lining cells of the small intestine will be studied in more detail at a later time.
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Locate the cells lining the lumen of the oviduct. Using the 20-40X, study the free surface of the
cells. The majority have fine, hair-like projections called cilia. At the apex of these cells note
the pink line, which indicates the presence of the basal bodies that give rise to the cilia.
Consult electron micrographs for the content and morphology of cilia and their basal bodies.
There are also secretory cells along this epithelium. These have elongated nuclei and
sometimes project above the epithelial surface
Uterine Tube
Epithelium
cilia secretory cell
basal bodies
nuclei
basement
membrane
MITOSIS
Mitosis is divided into four distinct stages: prophase, metaphase, anaphase, and telophase.
During prophase, the nuclear envelope disperses, replicated chromosomes condense, and the
two sister chromatids become attached at a site called the centromere. At metaphase,
duplicated chromosomes become aligned in a single plane. At anaphase A, the sister
chromatids separate and begin to migrate to the poles. At anaphase B, the sister chromatids
continue to migrate toward the poles and the microtubules of the spindle elongate. During
telophase, the sister chromatids reach the poles, the nuclear envelope re-forms and the
chromosomes decondense. Cytoplasmic division usually begins in anaphase and is complete
by the end of telophase.
The whitefish embryo has been stained with hematoxylin and eosin (H&E). There are
examples of cells at all stanges of the cell cycle since the cells are dividing asynchronously.
Assess nuclear envelope breakdown, chromosome condensation, mitotic spindle
development, and location of condensed chromosomes in the whitefish mitotic cells. On the
basis of these parameters, identify and determine the distinguishing features of cells in
prophase, metaphase, anaphase (A and B) and telophase.
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ELECTRON MICROGRAPHS
Examine the listed electron micrographs so that you understand the ultrastructural equivalents
of the structures you have seen at the LM level on the slides. Also use the electron
micrographs to examine organelles that are not visible at the LM level.
QUESTIONS
Answers to questions for all laboratories are given at the end of the laboratory manual, p. 86.
2. What is the relationship between heterochromatin and the synthetic activity of DNA?
6. How many membranes comprise the nuclear envelope? The wall of a mitochondrion?
7. What are the cytological and functional differences between cilia and microvilli?
EPITHELIUM
Learning objectives:
1. Recognize the different types of epithelia.
2. Relate characteristics of particular epithelia to their function, keeping in mind their essential
features including junctions, apical modifications, and polarity.
An epithelium is a layer or sheet of cells that covers a surface or lines a cavity. Functions of
epithelia include formation of a protective layer (epidermis), absorption of water and solutes
(intestine), secretion (intestine, various glands) and excretion (kidney tubules). Classification of
epithelia is generally based upon two criteria: number of cell layers and cell shape.
Simple epithelia are one cell layer thick and stratified epithelia are two or more cell layers
thick. Pseudostratified epithelium is an intermediate type that appears stratified but really is
one cell layer thick. The shape of epithelial cells may be squamous, cuboidal, or columnar;
intermediate forms are often encountered. Stratified epithelia are classified according to the
shape of the cells at the free surface and can be squamous, cuboidal, columnar, or
transitional. Transitional epithelia line cavities in the urinary tract, which may be distended, and
the thickness of the epithelium varies with the degree of distention.
Beneath the layer of epithelial cells is an underlying non-cellular structure known as the basal
lamina, which is secreted by the epithelial cells. The basal lamina is often associated with an
additional layer secreted by other cells. Together the basal lamina and the underlying layer
make up the basement membrane, which can usually be seen with light microscopy. Higher
magnification (e.g., electron microscopy) is usually required to resolve the basal lamina.
Epithelial Tissue
Types
Examine the epithelial cells. Note the basophilic structures that at the base of the cells are
rough endoplasmic reticulum. At the apex of the cell, secretory granules appear as acidophilic
structures. The contents of these granules are proteins, which are the precursors of digestive
enzymes. Review the subcellular structures involved in protein synthesis.
Examine the lining of the gall bladder as an example of simple columnar epithelium. Be sure
you locate regions where the epithelium is cut longitudinally to observe the simple columnar
epithelium. Note the microvillous border that you identified in the previous lab. In tangential
sections portions of cells in various planes of section may give the impression that the
epithelium is stratified. Why do the nuclei appear at different levels in tangential sections?
Locate the epithelium with its microvillous border and PAS positive glycocalyx. The basal
lamina is also PAS positive, but is not intensely stained. What is the basis for PAS stain?
In addition the Bodian silver stains secretory granules within enteroendocrine cells in the
epithelium and the basal lamina.
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PSEUDOSTRATIFIED EPITHELIUM
#5 Trachea, H&E
Pseudostratified epithelium appears to be stratified as nuclei are seen at various levels. In fact
all of the cells rest on the basal lamina, but not all of the cells have apices that reach the
lumen. The cells that are confined to the base are stem cells that are the sources of the cells
whose apices do reach the lumen.
Identify the two major types of cell that reach the lumen. What are their characteristics? What
is responsible for the eosinophilic line at the apex of the majority of the cells? Note the position
of the nuclei.
STRATIFIED EPITHELIUM
In this type of epithelium, no cells on the basal lamina reach the lumen. The layer of cells that
rests on the basal lamina is the source of the upper layers of cells.
#4 Skin, H&E
The epithelium of the skin is known as the epidermis. Its superficial layer is comprised of
keratinized (cornified) squamous cells. Note the multiple layers of the stratified squamous
epithelial layer of the skin. As cells are displaced toward the skin surface their characteristics
change. They ultimately die and are sloughed. This topmost keratinized layer stains dark pink
and has no nuclei because the cells are dead.
ELECTRON MICROGRAPHS
Examine the electron micrographs so that you understand the ultrastructural equivalents of the
structures you have seen on the slides.
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REVIEW
Reviewing what you have learned in the first labs, be sure that you know the structural
characteristics and functional significance of the following organelles and inclusions, and be
able to identify them in light and/or electron micrographs:
apical surface
microvilli
zonula occludens
golgi body
endoplasmic
reticulum
nucleolus
mitochondria
nucleus
basal lamina
basal surface
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CONNECTIVE TISSUE
Learning objectives:
1. Know that cells, fibers and ground substance constitute connective tissue.
2. Be able to describe the relationship of these constituents, their structures and functions.
3. Learn the distribution of collagen types (Types I, II, III and IV) in the connective
tissue types.
Connective tissue is comprised of cells, formed fibers, and amorphous extracellular matrix
(ground substance). Both the fibers and ground substance are secreted by the connective
tissue cells that are interspersed and embedded in the matrix. Functions of the connective
tissue include support and binding together of the other tissues; providing a medium for the
passage of metabolites; serving as a storage site for lipids, water and electrolytes; aiding in
protection against infection by an inflammatory reaction mediated by cells that have migrated
into the connective tissue from the blood; and repair by the formation of scar tissue.
Connective tissues are derived from the embryonic connective tissue or mesenchyme.
Mesenchyme is derived primarily from the mesodermal germ layer of the developing embryo,
but the ectodermal neural crest is known to give rise to some mesenchymal cells (ecto-
mesenchyme). See examples in subsequent lab (Cartilage, Bone, Bone Development).
CONNECTIVE TISSUE
This form of connective tissue has the largest number of cells per unit volume of extracellular
matrix. The large number of cells frequently makes it difficult to distinguish the fibrous
component without the use of special stains.
The fibers in the matrix have a loose and irregular arrangement, and they consist of
collagenous, elastic, or reticular fibers. Fibroblasts and macrophages are the most common
cells in loose connective tissue, but mast cells, plasma cells, neutrophils and fat cells may also
be found.
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Examine the scanned image at low power, and note that one surface is indented by pits that
are lined by columnar epithelial cells. Immediately beneath these cells is the loose connective
tissue called lamina propria.
Reticular tissue, a type of loose connective tissue in which reticular fibers are the most
prominent fibrous component, forms the supporting framework of the lymphoid organs (lymph
nodes, spleen, tonsils), bone marrow and liver. Reticular fibers (type III collagen) are too thin
to stain in ordinary histological preparations, but they are readily demonstrated by techniques
involving the reduction of silver from silver nitrate by the glycosaminoglycan surface coat.
#4 Skin, (H&E)
Under the stratified squamous epithelium examined earlier is the dense irregular connective
tissue of the dermis. Its thick collagenous (type I) bundles stain intensely with eosin and can
be seen to course in various directions.
Find the regions of the dense fibrous regularly arranged connective tissue (tendon). Collagen
is stained pink and can be distinguished from skeletal muscle that is stained purple. Note the
fibroblasts aligned along the collagen fibers in the tendon. These are flattened cells with
heterochromatic nuclei.
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Elastic
The fibers are predominantly elastic rather than collagenous. Elastic fibers stain reddish-brown
to black and form prominent fenestrated, elastic sheets in the aorta.
ELECTRON MICROGRAPHS
Examine the electron micrographs.
QUESTIONS
1. Are reticular fibers distinguishable in tissue stained with H&E?
2. Why do adipocytes appear empty?
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Learning objectives:
1. Describe the components and organization of cartilage and bone.
2. Relate the structure of bone and cartilage to their function.
3. Understand the differences between the development and growth of cartilage and bone.
4. Describe the processes of intramembranous bone development and endochondral
ossification.
CARTILAGE
Cartilage is the primary skeletal tissue of the fetus, and it serves as a model for the
development of endochondral bone. In the adult, cartilage forms the articular surfaces of joints,
the skeleton of the external ear, the septum of the nose, supporting rings and plates of the
trachea and bronchi, and intervertebral discs. Three types of cartilage are found in the adult:
hyaline, elastic, and fibrocartilage. These are classified according to the predominant
component of their extracellular matrix. As in other connective tissue classifications, there are
gradations between these basic types.
HYALINE CARTILAGE
#5 Trachea (H&E)
FIBROCARTILAGE
Fibrocartilage can be considered as a transitional type of tissue, between hyaline cartilage and
dense collagenous connective tissue, and it occurs in regions where support and great tensile
strength are desirable.
QUESTIONS:
1. What are the mechanisms of cartilage growth?
2. Are blood vessels found in cartilage and how does this relate to the nutrition of cartilage?
BONE
Bone is a calcified connective tissue, and like other connective tissues, it consists of cells,
fibers, and ground substance. The deposition of inorganic calcium phosphate salts as
hydroxyapatite crystals within its matrix is a distinguishing characteristic of bone. This renders
it structurally rigid. In addition, bone functions as a homeostatic reservoir of calcium and
phosphate ions and it encloses the hematopoietic elements of the bone marrow.
There are two types of mature bone, compact (lamellar) and spongy (trabecular or
cancellous). Compact bone is characterized by the regularity of its collagen fibers. Spongy
bone consists of a lattice of branching bony spicules, known as trabeculae, which are
surrounded by bone marrow in some regions. When the trabeculae are sufficiently thick, they
may contain osteons (see description below).
Immature (woven) bone (see below in "bone development") is the first bone laid down in
prenatal life or in the repair of bone fractures. In this type of bone, the matrix immediately
surrounding the osteoblast is called osteoid and is not mineralized. Immature bone is
characterized by irregularly arranged, interwoven collagenous fibers within a matrix containing
proteoglycans.
Because of its calcified matrix, bone presents difficulties in its preparation for microscopic
study. There are two basic techniques for studying bone with the light microscope, and both of
these types of preparations must be studied to appreciate the organic and inorganic
components of bone. (1) Bone may be decalcified by acid solutions prior to embedding and
sectioning. This permits study of the cells and organic matrix of the bone. (2) To study the
lamellar and canalicular pattern of the calcified matrix, it is necessary to grind down dried bone
that has not been decalcified to a thickness that permits the microscope light to be transmitted
(“ground bone”).
GROUND BONE
Cross and longitudinal sections (unstained). Use the illustrations in your textbook as a guide
and identify the following structures.
Haversian Systems (osteons) are distinctive structural units of compact bone that reflect the
developmental and nutritive pattern of its lamellar configuration. Haversian systems consist of
Haversian canals containing blood vessels and nerves surrounded by concentric lamellae of
bone. Lacunae lie between or within the lamellae. In life these lacunae are occupied by
osteocytes. Lacunae are connected with each other, and ultimately with the perivascular
spaces of the Haversian canal, by canaliculi. This communicating system of canaliculi is
essential for exchange of gases and metabolites between the osteocytes and the perivascular
spaces of the Haversian canal. Volkmann's canals, which also contain vessels and nerves, are
larger in diameter than Haversian canals and run perpendicularly to them.
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Interstitial lamellae lie between the more distinct Haversian systems; these are the remnants
of earlier Haversian systems that have been partially resorbed during bone remodeling.
A B C
DECALCIFIED BONE
This slide demonstrates periosteum, which has dense cortical bone on the surface (better
illustrated in the preceding slide) and spongy bone centrally. Osteoblasts are prominent on the
surface of the bony trabeculae. Osteoclasts (multinucleated giant cells with acidophilic
cytoplasm, related to the process of bone resorption) may also be seen near the
osteochondral junction. Calcifying cartilage and rows of hyaline cartilage cells are present and
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extend into the cartilage of the proximal end of rib. Around the rib section, skeletal muscle and
tendon are present.
Osteoblasts Osteoclast
QUESTIONS
Be sure you know how cartilage and bone differ morphologically, functionally, and with respect
to blood supply.
BONE DEVELOPMENT
The process by which bone is formed is termed osteogenesis or ossification. Bone is never
formed as a primary tissue, it always replaces a preexisting support tissue. There are two
types of bone formation: intramembranous and endochondral ossification.
INTRAMEMBRANOUS OSSIFICATION
#94 Parietal bones, Human fetuses, 3.5 and 6.5 mos. –Decalcified
At low power note the appearance of the developing bone as well as the total absence of
cartilage. In the bone from the older fetus, scalp is present in which numerous hair follicles can
be seen. Note the connective tissue has begun to condense as a fibrous periosteum on either
side of the anastomosing trabeculae of the growing bone. The trabeculae surround large
spaces (primitive marrow cavities) containing embryonic connective tissue, thin-walled blood
vessels, and nerves. In active regions, a unicellular row of osteoblasts (each with an eccentric
nucleus and strongly basophilic cytoplasm) lines the surface of the trabeculae. Osteoclasts
may be seen to occupy shallow pits in the bone (Howship's lacunae). Within the trabeculae,
notice osteocytes in their lacunae and the woven bone matrix, which, unlike that of mature
bone, is unevenly stained pink and exhibits a patchy basophilia. The acidophilic collagenous
fibers embedded in the matrix tend to be obscured by the matrix. At these stages the matrix is
not calcified (i.e., contains no calcium phosphate salts). This uncalcified early bone is termed
the osteoid. Later, minerals are deposited as minute hydroxyapatite crystals (calcium
phosphate salts) in close association with the collagenous fibers to form a solid rigid matrix.
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ENDOCHONDRAL OSSIFICATION
This is a longitudinal section cut through an interphalangeal joint. Locate the ends of two long
bones participating in the joint and identify the articular cartilage. Identify the epiphyseal disk,
the metaphysis, the marrow cavity, and the diaphyseal bone. In the epiphyses where growth in
length is occurring, note the zones of reserve cells, proliferation, maturation, hypertrophy,
calcification, ossification and resorption. What structure in mature bone is created by the zone
of resorption?
Each of these bones has a primary center of ossification. The zone of endochondral
ossification spreads from the primary ossification center toward the ends of the cartilage.
These slides do not show secondary ossification centers.
Note the bone of the diaphysis. Recall that this bone is growing in width by apposition and
remodeling along the periosteum and the endosteum. In the marrow cavity note the bony
spicules with calcified cartilage cores.
As the primary ossification center of the diaphysis advances toward the epiphyses, each
epiphyseal cartilage continues to grow and the whole cartilage model increases in length. This
increase in length and extension of the primary ossification center results in a sequence of
changes in the chondrocytes of the epiphyses, which is similar to that described for the
establishment of the primary center.
In the epiphyseal growth plate, observe the zones of reserve cells, proliferation, maturation,
hypertrophy, calcification, ossification and resorption.
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Secondary ossification centers have developed in the epiphyses. Enlargement of the epiphysis
occurs by growth of the articular cartilage. When growth ceases, the epiphyseal disk is entirely
replaced by spongy bone and marrow (“closure of the epiphyses”), resulting in a visible
epiphyseal line.
This slide includes a diarthrodial joint. In synovial or diarthrodial joints, articular cartilage caps
the ends of the bones, which are kept apart by a synovial cavity filled with synovial fluid. The
articulation is enclosed by a dense fibrous capsule, which is continuous with the periosteum
over the bones. Internal to this is the synovium, a secretory membrane formed by a layer of
collagenous fibers interspersed with flattened fibroblasts (synovial cells). This membrane is
commonly thrown into folds (synovial villi) that project into the synovial cavity.
ELECTRON MICROGRAPHS
QUESTIONS
NERVE
Learning objectives:
1. Know the components of tissue in the central and peripheral nervous systems.
2. Understand the meaning of terms gray matter and white matter.
3. Relate nerve function to the properties of neurons and their cell processes: axons and
dendrites.
The neuron is the structural and functional unit of the nervous system. Neurons are highly
polarized cells. The cell body contains the nucleus and synthetic apparatus. The highly
branched dendrites are the receptive pole and axons are the transmitting pole. Nervous tissue
is characterized by its extreme specialization for excitability and conductivity.
The supporting cells are in intimate contact with the neurons and their processes in both the
CNS and the PNS. These cells provide structural support and nutrients to the neurons. There
are also macrophages present in the nervous system; these cells are called microglia in the
CNS.
The connective tissue elements include the meninges, which surround the central nervous
system; capsules surround some sense organs and ganglia; and the endo-, peri-, and
epineurium of peripheral nerves.
Meninges
Dura mater
Arachnoid mater
Cerebral cortex
Pia mater
White matter
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The detailed structure and function of the nervous system will be studied during the
neurosciences course. The following class slides and electron micrographs will serve to
acquaint you with nervous tissue as one of the four basic tissues.
The central nervous system consists of the brain and spinal cord.
Spinal cord
Within the white matter, note the nuclei of glial cells (mostly
oligodendroglia) and the cross sections of axons (unstained).
The clear space surrounding each axon is occupied in life by
the myelin sheath.
Note the meninges surrounding the spinal cord. What are the
three layers of meninges? Is there anything inside the central
canal?
At low power identify the centrally located butterfly-shaped arrangement of the gray matter.
Within the gray matter, locate the cell bodies of neurons and the associated dendrites and
axons. Surrounding the gray matter is the paler staining white matter and the supporting cells
(oligodendroglia and others). In the gray matter, note the size and shape of the cell body of the
neurons, particularly those in the anterior (ventral) horn.
With the Cajal technique, silver is precipitated on neurofilaments within neuronal cell bodies
and their processes. In general, all of the cells and their processes are revealed by this
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technique. Note that in the gray matter most of the axons are oriented in the plane of the
section. In contrast, most of the axons of the white matter are viewed in cross section, since
the fibers are running to and from the brain and other segmental levels of the spinal cord. Note
the surrounding meninges, and identify the central canal of the spinal cord.
Brain
Observe the pale staining branches of the central white matter surrounded by a darkly stained
cortex. Identify the outer, pale-staining molecular layer of the cerebellar cortex, and the inner,
basophilic granular layer of the cortex. Both the molecular layer and granular layer constitute
the gray matter. The molecular layer contains axons and dendrites, but relatively few neurons
compared to the granular layer.
On these sections of the cerebellum, the cut surfaces may result in the exposure of the pale-
staining medulla (white matter) at the surface of the section, where it could be confused with
the molecular layer of the cortex. Try to find a surface covered by the meninges, to insure that
you are indeed looking at the cortical surface.
The peripheral nervous system includes all neural tissue other than the brain and spinal cord.
Preganglionic axons in both sympathetic and parasympathetic systems are myelinated while
postganglionics are unmyelinated. What is the functional consequence of the location of
parasympathetic neurons near the target organ?
The enteric nervous system is the intrinsic innervation of the gastrointestinal tract. It is made
up of ganglia and the nerves emanating from these neurons. It contains glia like the central
nervous system, however there are no Schwann cells, fibroblasts or other connective tissue
elements within the ganglia.
PERIPHERAL NERVES
In the cross section note the axon (black), which is surrounded in turn
by a myelin sheath and its Schwann cell neurilemma (brown). Locate
lightly myelinated and unmyelinated fibers. Speed of conduction is
related to the diameter (including myelin sheath) of a nerve fiber.
Examine the longitudinal and cross-section and identify the connective tissue of the
perineurium. Delicate reticular connective tissue, the endoneurium surrounds individual axons.
The epineurium, which is dense irregularly arranged connective tissue, binds many nerve
bundles. The epineurium is not present on these sections.
Identify the cross section of peripheral nerve. Note that the nervous tissue has shrunk within
the perineurium during tissue processing. Only some of the axons within the nerve have been
cut in true cross section. What are the cells within the nerve whose nuclei are stained? Find a
small nerve elsewhere in the tissue.
ELECTRON MICROGRAPHS
Examine the electron micrographs so that you understand the ultrastructural equivalents of the
structures you have seen at the light microscopic level.
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MUSCLE
Learning objectives:
1. Be able to distinguish the three types of muscle.
2. Be able to describe how they differ in structure and function.
Muscle is especially adapted for contractility with elongated cells arranged in parallel to the
direction of contraction. This contraction is used to move the body or change the shape of
certain organs. Blood vessels within the associated connective tissue supply a rich blood
supply to provide nutrients and oxygen and to eliminate waste products. Nerves also
accompany the blood vessels in the connective tissue.
The unit of muscle tissue is a cell, often referred to as a muscle fiber. The term "fiber" is used
here in contrast to a connective tissue fiber, which is non-cellular, and to a nerve fiber which is
a cell process. Every muscle fiber is surrounded by a basal lamina called the endomysium. Its
plasma membrane (which is not visible with the light microscope) is often called the
sarcolemma and its cytoplasm is given a special name, sarcoplasm. Within the sarcoplasm
are cytoplasmic contractile elements, the myofilaments.
On both a structural and functional basis, muscle is classified as smooth, skeletal or cardiac.
Skeletal and cardiac muscle fibers have a characteristic striated appearance due to the
organization of myofilaments. In smooth muscle fibers the myofilaments are not arranged with
regularity and so these cells are nonstriated.
SKELETAL MUSCLE
Skeletal muscle is composed of large, cylindrical, multinucleated cells. The most striking
feature of skeletal muscle fibers is the presence of striations, which are visible in longitudinally
sectioned fibers. The striations are due to the presence of myofibrils, which are cylindrical
bundles of thick and thin myofilaments, organized into units of contraction called sarcomeres.
The orderly arrangement of these repeating units within the myofibrils gives rise to the
characteristic pattern of transverse banding.
At low power examine bundles of skeletal muscle fibers and sheets of tendon (dense regular
connective tissue). Under higher magnification, skeletal muscle fibers may be recognized by
their cross-striations. In this preparation most tendon has a homogenous, almost glassy,
appearance (this is a diagnostic feature). The cells of the tendon (fibroblasts or fibrocytes)
occur in rows, squeezed between the thick collagenous fibers; only their flattened, rod-like
basophilic nuclei show well. The zone of insertion of the skeletal muscle into the tendon is
obvious, but higher resolution with the electron microscope is necessary to see the detailed
structure of the junction.
Define a sarcomere. Be sure you know what the electron microscope has revealed about its
fine structure. Know the structural changes that occur in a sarcomere during contraction and
the theory that has evolved from electron microscopic studies to explain muscle contraction.
CARDIAC MUSCLE
Cardiac muscle is striated and contains centrally placed nuclei. Cardiac muscle cells are
branched cylinders connected by intercalated discs.
FYI: Intercalated disks have both transverse components comprised of fascia adherens
and desmosomes and longitudinal components containing mainly gap junctions.
32
SMOOTH MUSCLE
Smooth muscle is widely distributed in the body. It is found in the walls of ducts and blood and
lymphatic vessels, as well as in the walls of the digestive, respiratory and urogenital tracts. It
also occurs in many other sites including the eye (iris and ciliary body), skin (arrector pili
muscles of hairs), endocardium, scrotum, penis, perineum, and nipple. Always correlate the
function of smooth muscle with the different organs and regions in which it is found.
At low power identify the broad expanse of smooth muscle (the myometrium). Under higher
power, note that the fascicles of smooth muscle are arranged in various planes. There are
numerous blood vessels within the myometrium. The larger muscular arteries when cut in
cross section appear as swirls of smooth muscle.
NOTE: Smooth muscle and connective tissue both stain pink with H&E. To distinguish
between these two types of tissue special stains such as Mallory trichrome can be used. With
the Mallory stain collagen stain blue and smooth muscle fibers stains.
Using higher magnification, examine the muscle layers and notice that the musculature is
skeletal mixed with bundles of smooth muscle fibers due to the level of the esophagus at
which this section was taken. In the skeletal muscle, note the strong acidophilia and peripheral
nuclei. In longitudinal sections identify cross-striations. In cross-section note that the skeletal
muscle fibers resemble rounded polygons separated by the endomysium with the nuclei
clearly visible the periphery.
ELECTRON MICROGRAPHS
QUESTIONS
1. Why do smooth muscle fibers in cross section have different diameters and why do some
of these fail to show nuclei?
2. Are myofibrils or sarcomeres present in smooth muscle fibers?
35
Learning objectives:
1. In Wright’s stained blood smear:
a. recognize red cell morphology.
b. distinguish types of leukocytes.
c. be aware of the presence of platelets.
2. In smear of bone marrow:
a. recognize stages of erythropoiesis.
b. recognize stages of granulopoiesis.
c. identify megakaryocytes.
3. In section of bone marrow:
a. Understand the structure of the tissue.
b. Recognize megakaryocytes.
c. Be aware of the presence of fat.
BLOOD
The study of normal and pathologic blood development is based on the examination of stained
smears of peripheral blood and bone marrow and sections of lymphoid tissue of the thymus,
spleen, lymph nodes and lymphoid infiltrations and aggregates along the G.I. tract. During this
lab, become familiar with the morphology of mature peripheral blood cells and study the
stages of blood development.
The cellular or formed elements of peripheral blood are classified according to the details of
their appearance following staining with polychromic stains, e.g. Wright's stain. Four groups or
classes of cells or cell fragments are usually present in peripheral blood:
To study the cell types in your preparations first scan the smear at low power and select a
region where the red cells do not overlie one another and where they are stained pink. After
finding an area that is well smeared and well stained, use the highest magnification to examine
the blood cells, particularly the leukocytes. Identify neutrophils, lymphocytes, eosinophils, and
monocytes. This slide does not have basophils, which account for less than one percent of the
total number of leukocytes. Platelets will also be found. Use the chart and images on the
following page to assist in identifying these different cell types.
36
Gartner & Hiatt Color Atlas of Histology 4th ed., Lippincott Williams & Wilkins, 2006
neutrophil
erythrocyte monocyte
basophil
platelets
lymphocyte
eosinophil
37
HEMATOPOIESIS
BONE MARROW
The marrow of adult human bones is the major site of formation of erythrocytes, granulocytes,
monocytes and platelets. Some lymphocytes are formed in the bone marrow as well.
Lymphocytes will be studied in more detail with lymphoid tissue.
There are stem cells that are precursors of both the red and white blood cell series, however
because their proportion in bone marrow is low it is unlikely that you will be able to recognize
them in your slides. Do not spend time looking for stem cells.
The student should bear in mind that the frequency of any given cell type or stage of
differentiation in the marrow is a complex function related to the relative frequency of the cells
in the blood, to the half life of the cell type in the circulation, and to the maturation time of the
cells in the marrow. The most frequent precursor series is the erythroid cell series, followed
closely by the neutrophilic series.
Distinctions between the cells of the granulocytic series are based on nuclear morphology and
the size and staining properties of the granules. High magnification is required for this type of
determination. Criteria for evaluating cell type and stage of maturation are as follows:
Size of the cell and its nucleus: In general there is a gradual decrease in size of the cell and its
nucleus with maturation. However, remember that cells preparing to undergo mitosis enlarge
before division. Also some cells may be flattened more than others during preparation so size
alone can be misleading.
Appearance of the chromatin network in the nucleus: Immature cells have a delicate fine-
meshed chromatin network. More mature cells have coarser, more condensed chromatin.
Presence or absence of nucleoli: Nucleoli are visible as pale blue circular areas within the
chromatin network of immature cells.
Cytoplasmic basophilia: Very immature cells have pale blue cytoplasm, due to the presence of
only a few scattered ribosomes.
Specialized cell products: Accumulation of hemoglobin in erythroid cells, and the appearance
of granules and their type in granulocytes.
ERYTHROID SERIES:
Basophilic erythroblast - This cell is usually smaller and the nucleus, which is
intensely heterochromatic, is centrally located. The cytoplasm is a deeper blue
color than that of the proerythroblast, due to the mixture of abundant free
ribosomes and the initiation of hemoglobin synthesis. There is no longer a
visible Golgi apparatus.
amount of hemoglobin being synthesized by the ribosomes. This is the last stage during which
cell division occurs.
Erythrocyte (RBC) - The extrusion of the nucleus from normoblasts results in the formation of
anucleate erythrocytes. Occasionally there is still some residual basophilia in the cytoplasm of
these cells, due to the retention of some ribosomes. Such immature red cells are called
reticulocytes because of the so-called reticulated pattern of cytoplasmic basophilia. Under
normal conditions, a small percentage of reticulocytes enter the circulation before completing
their maturation. However, when there is a great increase in erythrocyte production the
percentage of reticulocytes entering the blood increases. Reticulocyte counts can provide
information about the rate of erythrocyte production.
GRANULOCYTIC SERIES:
Myeloblasts are stem cells that differentiate into the granulocytic series. The myeloblast is a
large cell with a large ovoid pale-staining nucleus, 2 to 5 nucleoli, and lightly basophilic
cytoplasm (due to a scattering of ribosomes). These cells are difficult to distinguish.
The textbook should be reviewed before an attempt is made to identify the precursor stages of
myeloid differentiation on this slide. Begin by scanning the slide under low magnification. The
most immediately obvious cell type will be the enormous megakaryocyte which gives rise to
blood platelets. Choose an area of this slide where the cells are not too closely smeared to
study the cells of the erythroid and granulocyte series.
39
The following points should be kept in mind when examining bone marrow smears:
2. You should be able to assign a well-fixed and well-stained cell to either the erythrocyte
or granulocyte line of development.
3. You should be able to say whether a cell is relatively undifferentiated (i.e., not far
removed from the stem cell) or nearly, or completely, differentiated.
4. If a cell in the granulocyte line already has specific granules it should be further
classified into the neutrophilic, eosinophilic, or basophilic series and based on its
nuclear morphology, whether it is a myelocyte, metamyelocyte, or a mature cell.
Be sure you know the biochemical composition of the cytoplasmic granules of neutrophils
(polymorphonuclear leukocytes), eosinophils, and basophils.
This is a bone marrow core biopsy. Note the spicules of bone and
intervening marrow. The marrow is approximately 60-70% cells and 30-40%
fat. Note the conspicuous megakaryocytes and areas of erythroid and
myeloid development.
Like the section of human bone marrow, this also reveals the natural relationship of developing
blood cells to one another and to sinusoids. Note the megakaryocytes and the developing red
and white blood cells. The cells of the erythroid and myeloid series tend to be grouped in small
foci and that within each group the cells tend to be at the same stage of development.
Variable amounts of adipose tissue are present.
ELECTRON MICROGRAPHS
QUESTIONS
1. What is the functional significance of the cytoplasmic staining affinities of the basophilic
erythroblast, polychromatophilic erythroblast, normoblast and erythrocyte?
LYMPHOID TISSUES
Learning objectives:
1. Know the structure and function of the lymphoid tissues and organs: (including afferent and
efferent flow and specialized vasculature)
a. bone marrow
b. thymus
c. tonsil
d. diffuse lymphoid tissue (BALT and MALT)
e. lymph nodes
f. spleen
The lymphoid system has primary lymphoid organs that produce lymphocytes and secondary
lymphoid organs which are the sites of immune response.
Primary lymphoid organs include bone marrow and thymus. All lymphocytes originate in the
bone marrow. B cells (as well as monocytes, erythrocytes, granulocytes, and megakaryoctyes)
remain in the bone marrow as they mature. T cells migrate to the thymus before maturation.
Secondary lymphoid organs include tonsil, lymph nodes, spleen, and diffuse lymphoid tissue
(bronchial-associated lymphoid tissue or BALT in the lungs and mucosa-associated lymphoid
tissue or MALT in the digestive tract).
Lymphatic vessels carry lymph fluid, which contains particulate matter and protein that escape
from blood capillaries as well as ingested fats, microorganisms, and other antigenic material
that penetrate epithelial surfaces. Lymph is filtered in lymph nodes and then returned to the
circulation via the thoracic duct and the right lymphatic duct.
TONSILS
The tonsillar ring is located near the entrance of the throat and consists of the palatine tonsil
(commonly known as "the tonsil"), the pharyngeal tonsil (commonly known as "adenoids”), and
the lingual tonsil (on the posterior surface of the tongue)
This is a section through the palatine tonsil. Notice the stratified squamous non-keratinized
epithelium covering the free oropharyngeal surface of the tonsil. In the underlying lamina
propria identify simple and branched epithelial crypts sectioned in different planes and
representing tubular invaginations of the surface epithelium. The lining epithelium of the crypts
may show evidence of keratinization or erosion and can be obscured when heavily infiltrated
with lymphocytes.
individual crypt as an axis. At one side of the section in the submucosa, note the presence of a
pure mucous gland.
Within the lamina propria identify lymphatic nodules and note germinal centers of the active
antigenic response.
LYMPH NODES
afferent lymph
medullary sinus
trabeculae medullary cord
artery
capsule
vein
subcapsular sinus
efferent lymph
lymphoid nodule
4. Connective tissue trabeculae, stained red-brown and extending from the capsule into the
cortical region.
5. The cortex, composed of lymphatic tissue with nodules (poorly defined in this node).
6. The paracortex, the non-nodular region of the cortex
7. The medulla, characterized by lymphoid tissue arranged in branching and anastomosing
medullary cords and medullary sinuses.
8. An indented region, the hilum, which is surrounded by the medulla and contains connective
tissue, a few fat cells, blood vessels, nerve bundles and large efferent lymphatic vessels
with delicate valves to prevent the backup of lymph into the node. (The scanned section
does not pass directly through the site of the hilum but there is a large the efferent
lymphatic vessel in the center of the medulla.)
Be sure you understand the cellular interactions and activities within both the cortical and
medullary regions of the lymph node.
#24 Lymph node. injected with India ink through lymphatic system
Under low power, notice that the India ink particles are concentrated mainly in the sinuses of
the node and to a lesser extent in the medullary cords, where most of the phagocytic cells are
located. The ink is virtually absent in the cortical nodules. At higher magnifications, the ink
particles may be seen within the macrophages. The subcapsular sinus is clinically important
because neoplastic cells enter the lymph node here. On the convex surface of the node
afferent lymphatic vessels with valves may be seen.
THYMUS
The thymus is the site of T-cell maturation. Thymic epithelial reticular cells attract thymocyte
precursors and macrophages. The functional blood-thymus barrier consists of epithelial
reticular cells, their basal laminae, and endothelial cells joined by tight junctions. This barrier
keeps antigens in blood vessels from entering the thymus, preventing reaction with developing
T-cells. The blood-thymus barrier is present in the cortex, but not the medulla.
This slide shows a section passing through a portion of one thymic lobe, surrounded by its thin
connective tissue capsule. Thinner connective tissue partitions extend from the capsule and
divide the thymic parenchyma incompletely into many angular thymic lobules, most of which
are characterized by a peripheral dark cortex and a central paler medulla. The medullary
tissue forms a continuous branching central core within each lobe.
At higher magnification, the cortex may be seen as a dense layer of closely packed cells,
mainly thymocytes. The fairly sharp demarcation of heavily stained small thymocytes in cortex
is more obvious than in the medulla. It is the round nuclei of these small thymocytes with very
condensed chromatin that impart to the cortex a deeply stained appearance in this H&E
preparation.
NOTE: The normal thymus lacks both lymphatic nodules and lymphatic or blood sinuses.
Unlike lymph nodes, the thymus is not interposed in the lymph circulation and has no afferent
lymphatic vessels.
44
Compare this section with slide #26. Involutional changes are evident in this section of adult
thymus. As seen on the preceding slide, in childhood (from birth to 10 years of age) the
thymus consists of closely crowded lobules of thymic tissue with thin connective tissue capsule
and septa. At puberty (from about 11 to 15 years), the thymic parenchyma remains prominent
but the interlobular septa become broader. Then the thymus begins to decrease in size, fat
begins to appear, and changes known as "age involution" occur. From about 21 to 45 years,
the adipose tissue becomes increasingly prominent and occupies a larger area than the
parenchyma of the thymus. Notice that the cortex has lost density and the cortico-medullary
boundary is obscured. The framework of epithelial-reticular cells of the cortex has collapsed
and the cortical thymocytes have decreased in number, however the medulla may be seen to
have suffered little change and large Hassall's corpuscles can still be readily identified. In older
individuals, Hassall's corpuscles appear to be fewer in number.
NOTE: Despite post-pubertal involution, the thymus remains a functional organ with
recognizable cortex and medullary regions throughout life. The thymus may also undergo
"accidental" or "stress involution" due to chronic illness.
SPLEEN
#76 Spleen
The spleen is comprised of red pulp and white pulp. The red pulp is the site of blood filtration
and the white pulp is lymphoid tissue that responds to blood-borne antigens.
Identify under low power some of the structures that are diagnostic of the organ. There is a
dense connective tissue capsule that sends conspicuous trabeculae to partially subdivide the
organ. Unlike the thymus and lymph nodes, the spleen lacks division into cortex and medulla.
The remainder of the spleen consists of red pulp and is composed of sinusoids (modified
blood vessels) and splenic cords (of Billroth). The latter are cellular regions organized as
plates of loose lymphatic tissue separating the sinusoids. It is not always possible to
distinguish Billroth cords from the sinusoids, as is evident in this preparation where the
sinusoids are partially collapsed. Under higher magnification, look for transverse and
longitudinal sections of patent sinusoids. The lining cells of these sinusoids are elongated
endothelial cells with tapered ends that lie parallel to the long axis of the vessel. These
endothelial cells are separated from each other by gaps. In cross sections of sinusoids,
therefore, the lining reticular cells are cut transversely and appear as cuboidal blocks arranged
loosely in a circle, with intervening gaps.
Like the PAS technique, this method stains the network of reticular fibers and the fenestrated
basal laminae of the splenic sinusoids black. In section, the membrane may be seen as a
succession of black points or short lines of silver-impregnated substance.
ELECTRON MICROGRAPHS
QUESTIONS
CARDIOVASCULAR SYSTEM
Learning objectives:
1. Understand the structure of the heart and its conducting system.
2. Know the components of the vascular wall and how these differ among the types of
blood vessels.
The cardiovascular system is composed of the heart and a continuous system of blood
vessels including arteries, arterioles, capillaries, venules, and veins. Together the lymphatic
vessels and the cardiovascular system form the circulatory system.
THE HEART
Use the image below as well as illustrations of the heart in your gross anatomy textbook or
atlas to help you to orient your slide and to locate the atrium, ventricle and mitral valve.
Valve
Ventricle
Atrium
fascicles are longitudinally sectioned, note the intercalated discs which appear as red-staining
step-like lines perpendicular to the long axis of the fiber. Is the myocardium thicker in the
atrium or ventricle? Why?
The heart valves are extensions of the innermost layer, the endocardium. The endocardium
contains an endothelium on the free surface and underlying supportive connective tissue. Use
this slide to also study the structure of the arteries and veins of the coronary circulation.
BLOOD VESSELS
2. Tunica media. This layer is composed predominantly of circularly arranged smooth muscle
fibers. There may also be a variable amount of reticular and elastic fibers.
3. Tunica adventitia. This coat consists predominantly of fibroelastic connective tissue whose
fibers generally occur in a longitudinal array. In larger muscular arteries, there is frequently
an external elastic membrane separating the tunica adventitia from the tunica media.
DISTINGUISHING FEATURES
4. Capillaries are the easiest vessels to define (but not to find). They
consist of an endothelial layer and its underlying basal lamina.
There may also be an associated pericyte within the basal lamina
of the endothelial cell. They are classified on the basis of their
“leakiness” as continuous (e.g., muscle, central nervous system,
lungs), fenestrated (e.g., endocrine glands, intestinal tract, gall
bladder), and discontinuous or sinusoidal (e.g., spleen, bone
marrow, liver).
49
Types of Capillaries
The Aorta
The sections on these slides are stained to demonstrate elastin, collagen and the cellular
organization of the aorta. The aorta is an elastic artery which has a relatively thick tunica
intima bounded by endothelium and the internal elastic membrane. The internal elastic
membrane, however, is less obvious here than in the smaller muscular arteries. In the tunica
intima smooth muscle cells run parallel to the long axis of the aorta while in the tunica media
smooth muscle is spirally arranged. Within the tunica media the distribution of elastin in the
elastic laminae is revealed as red-staining or black-staining material by the elastin stain.
Elastin is not stained in the Masson preparations, but can still be seen as clear, refractile
material surrounded by blue-staining collagen fibers. Both elastin and collagen are produced
by smooth muscle cells, which are the only cell type within the tunica media.
In slides #16 and #20 the blood vessels supply the aorta, the vasa vasorum, should be
identified in the tunica adventitia.
In addition to locating blood vessels, also observe the numerous sectioned nerves and
adipose tissue. Note the presence of brown fat cells with their multilocular appearance.
50
Medium and small size arteries and veins occur in most of your slides, and they will be seen
during the study of every tissue and organ. The following slides are particularly useful for
distinguishing arteries and veins.
Identify large and small veins and venules, large and small arteries and arterioles. Compare
the tunica media and the tunica adventitia in these vessel types. Note that in arteries and
arterioles there is an internal elastic membrane. This appears wavy due to the contraction of
the smooth muscle that underlies it. Identify capillaries and know their distinguishing
characteristics.
Use any (or all) of the following slides to distinguish arteries, arterioles, veins and capillaries.
#4 Skin
Note the presence of small blood vessels and sweat glands with
ducts in the dermis. Blood vessels have an endothelium,
whereas sweat glands and ducts are lined by cuboidal
epithelium.
Longitudinally
#50 Kidney sectioned capillary.
Capillaries can easily be distinguished in the white fat in the renal pelvis.
ELECTRON MICROGRAPHS
QUESTIONS
2. Examine the drawings on the front and the back of this manual. What types of capillaries
are these? Where might such capillaries be found?
51
SKIN
Learning objectives:
1. Know the layers of the skin.
2. Understand how the components of the skin serve its various functions.
This laboratory exercise serves both as an introduction to the skin, the largest organ of the
body, and as a review of the major tissues. As you study the slides of the skin, identify
examples of epithelium, connective tissue, muscle and nerve.
The majority of skin is thin skin, most of which is hairy. Thick skin is restricted to the ventral
surface of the hands and feet. All skin is made up of three layers:
Epidermis:
The stratified squamous keratinizing epithelium of the epidermis is made up primarily of
keratinocytes. The form and function of these cells changes as they pass from basal to
superficial locations. The layers of the epidermis from basement membrane to skin surface
include:
Stratum basale: Cells of all the layers are generated from the keratinocytes in this layer.
Therefore, you may see mitotic figures. The keratinocytes in this and the overlying layers
contain melanin granules that have been transferred to them by melanocytes. The cytoplasm
of melanocytes does not stain with H&E, giving the appearance of a halo. Special staining
methods are required to identify melanocytes definitively.
Stratum spinosum: This is several cell layers in thickness. The cells are attached to each
other by intercellular bridges (desmosomes). Because the cells pull apart during preparation,
the attachment sites give the cells a spiny appearance.
Dermis:
Papillary layer: loose connective tissue underlying the basal layer of the epidermis containing
blood vessels, nerves, and lymphatic vessels. Dermal papillae may contain sensory nerve
endings called Meissner’s corpuscles.
This is loose connective tissue containing abundant adipose tissue. It is a good region to
examine glands, ducts, blood vessels and nerves.
Eccrine sweat glands are present in high concentration in the dermis and subcutaneous
tissue. They are coiled tubular glands with an acidophilic margin, which corresponds to the
layer of myoepithelium. The ducts are straight as they lead through the superficial dermis to
the basal aspect of the epidermis. At this point they assume a coiled pathway, which becomes
corkscrew-like in the stratum corneum.
Pacinian corpuscles are another type of nerve ending found in the dermis or subcutaneous
tissue. They are made up of an axon surrounded by numerous concentric cellular lamellae.
These are also found in other regions of the body.
Hair follicles are well demonstrated in this slide in longitudinal section. The thin epidermis,
characteristic of hairy regions, has a lacy or frayed stratum corneum whose appearance is an
artifact of sectioning. In life, this layer of the epidermis would be more compact and only the
most superficial keratinized cells would be desquamating.
ELECTRON MICROGRAPHS
QUESTIONS
RESPIRATORY SYSTEM
Learning objectives:
1. Recognize major parts of the respiratory track: trachea, bronchi, bronchioles, terminal
bronchioles, respiratory bronchioles, alveolar duct, and alveoli.
2. Understand the blood-air interface in the alveolus and know the function of the type I and II
pneumocytes and alveolar macrophage.
The respiratory system functions in the exchange of gases between the external and internal
environments. Major parts of the system include the nasal cavity, larynx, trachea, bronchi, and
pulmonary alveoli. Some regions of the respiratory system are specialized for conduction of
gases and other areas function primarily in the exchange of gases. Filtration mechanisms exist
in both regions. The function of each part of the respiratory system is reflected in the structure
of its wall: the type of epithelium and its apical modifications, intraepithelial and subepithelial
glands, cartilaginous rings and plates, smooth muscle and elastic tissue, and the relationship
of capillary endothelium to alveolar epithelium.
Segment Characteristics
#5 Trachea
In this cross section of the trachea note the C-shaped ring of hyaline cartilage and the
pseudostratified ciliated columnar epithelium. Cilia are clearly seen in some areas but goblet
cells may not be obvious. Exocrine glands are found beneath the epithelium. Lymph nodes
and diffuse, sub-epithelial lymphatic tissue are seen. Note also the nerves, blood vessels and
adipose tissue. This is a good slide to review these structures.
Look for the same structures in this slide and the following slide. What criteria do you use to
distinguish between the esophagus and the trachea?
55
Be sure you know the types of cells found in the alveolus and
how they can be distinguished structurally. Know the function of each cell type. Use TEMs to
identify cell types and to aid in understanding the structure of the alveolar wall.
This stain highlights the elastic fibers (red brown) in the lung.
ELECTRON MICROGRAPHS
QUESTIONS
5. Which cells are responsible from keeping the lungs free from obstructing particulate
matter? How do they carry out this function?
57
URINARY SYSTEM
Learning objectives:
1. Be familiar with the organization of the urinary system, including the kidney, ureter,
bladder, and urethra.
2. Understand the organization of the vascular and urinary parts of the kidney.
3. Recognize the different parts of the nephron and be able to describe their structure and
function
The urinary system consists of the kidneys, ureters, urinary bladder, and the urethra. The
kidney is specialized for the removal of waste products from the blood and for the regulation of
water and salt balance of the blood and intercellular fluids.
The kidney is divided into lobes. One lobe consists of the conical medullary pyramid and the
cortical substance that surrounds it like a cap. This slide demonstrates a unilobar kidney. The
human kidney is composed of 12-13 lobes.
Identify the outer, brighter staining cortex and central, paler staining medulla. The cortex is
characterized by round capillary tufts, called glomeruli, within the renal corpuscles. The base
of the medullary pyramid lies below the cortex, and the apex of the pyramid projects or
empties into the renal pelvis. The hilum of the kidney is the site of entrance and exit of the
renal artery, vein, and ureter. Note the abundance of white fat in this region.
Examine the junction between cortex and medulla. This junction is irregular. The cortex is
subdivided into alternating regions: 1) the cortical labyrinth consisting of glomeruli and
convoluted tubules and 2) the medullary rays consisting primarily of radially directed straight
segments of the loop of Henle and collecting tubules. A kidney lobule consists of a medullary
ray and the portions of the adjacent cortical labyrinth. The medulla is further sub-divided into
an outer zone adjacent to the cortex and an inner zone including the tip of the pyramid (which
is called the papilla).
With medium power identify the different regions of the nephron, the structural and functional
unit of the kidney. A nephron is composed of: 1) renal corpuscle, consisting of the vascular
glomerulus and its capsule (Bowman's capsule); 2) proximal convoluted tubule; 3) loop of
Henle, consisting of a thick descending segment, a thin U-shaped segment, and a thick
ascending segment; and 4) distal convoluted tubule. The excretory portion of the kidney
begins with the collecting tubules (which are in continuity with the distal convoluted tubules).
Next, examine the medullary rays adjacent to the labyrinth and the
medulla itself.
Identify the collecting tubules in the rays and in the medulla. These latter tubules are pale-
staining like the distal tubules, but differ from them in that their epithelium is more columnar,
the apex of the epithelial cells tend to bulge into the tubule lumen, and the intercellular
boundaries are readily evident as the cells do not form interdigitations.
1. The radially running thick descending segment of the loop of Henle (cytologically similar in
appearance to the proximal convoluted tubules with which they are continuous in the ray)
2. The thin segment of the loop of Henle (in most cases the simple squamous epithelium of
these tubules cannot be distinguished from that of a capillary in the inner zone of the
medulla)
3. The thick ascending segment of the loop of Henle (cytologically similar in appearance to
the distal convoluted tubules with which they are continuous) returns to the glomerulus of
origin of the nephron and forms the macula densa (see description above)
Try to visualize the spatial relationships of an entire nephron as you examine the cortical
labyrinth and rays, and consider which components you would expect to find in each region.
Orient yourself as with the previous slide, and then examine the cortical labyrinth with medium
power. Locate a region with several renal corpuscles. The staining differences of the general
cytoplasm in the two types of convoluted tubules are not as distinct with this stain as with H&E,
but the proximal convoluted tubules can be readily identified by the PAS-positive brush border
59
Examine electron micrographs of the glomeruli, proximal and distal convoluted tubules in your
textbook and in the lab, and correlate the PAS-positive structures evident with the light
microscope with their ultrastructural counterparts. What is the functional significance of the
occurrence of a brush border in the proximal tubule? Be sure you understand the significance
of PAS staining.
Examine the cortical labyrinth and rays as described previously. Be certain that you
understand the blood supply of the renal corpuscle, the convoluted tubules, and the loop of
Henle, and the functional significance of these. Consult your textbook and its illustrations.
What are the components of the arterial portal system of the kidney?
URETER
Be certain that you can recognize transitional epithelium in both the relaxed and expanded
states. The basic structural arrangement of the bladder is similar to that of the ureter, and the
structures and layers should be studied as in the previous slide. Note the greatly thickened
muscularis of the bladder. This slide shows the bladder in the relaxed state.
60
URETHRA
This tissue is readily identified at lowest magnification by the characteristic shape of the lumen
of the penile urethra and by the surrounding sponge-like arrangement of erectile tissue filled
with blood. Study the epithelial lining of the penile urethra and observe that its appearance
varies in different regions of the same section. Most frequently it appears to be
pseudostratified or stratified columnar. Mucous glands may occur as nests of epithelial cells
along the lining epithelium (intra-epithelial glands) or they may occur as more typical urethral
glands (of Littre) whose ducts empty into local recesses of the urethral lumen.
The erectile tissue and the supporting fibro-muscular network of trabeculae that supports them
are considered in the chapters on the male reproductive system.
ELECTRON MICROGRAPHS
QUESTION
ENDOCRINE GLANDS
Learning objectives:
1. Be able to recognize each of the endocrine organs and relate their structure to function. Be
aware of the close proximity of the vasculature in the endocrine organs.
2. Know the products of the organs and how their synthesis and release are controlled.
PITUITARY GLAND
The pituitary gland is a dual gland consisting of an epithelial component called the
adenohypophysis and a neural component called the neurohypophysis. The adenohypophysis
is derived from an outgrowth of oral ectoderm known as Rathke’s pouch. It has three parts, the
pars distalis (anterior lobe), pars tuberalis (enveloping the infundibular stalk), and pars
intermedia (rudimentary in adults). The neurohypophysis is a neuroectodermal downgrowth
from the floor of the diencephalon (part of the central nervous system) and includes the pars
nervosa (posterior lobe) and the infundibulum.
With Masson's stain, the acidophils are red and the basophils are blue. Chromophobes will be
light orange or faded to grey. Note that red blood cells may be anything from red to blue. The
cords and clumps of epithelial cells are sharply outlined by the blue collagenous fibers.
The red or blue staining of the secretory granules is due to the acidophilia or basophilia of the
hormone contained in the granules. The pars intermedia, which is not seen clearly on this
slide, forms a cap around the neurohypophysis and separates it from the pars distalis. What
kinds of cells are in the neurohypophysis?
Nuclei are purple-black, acidophils bright pink, basophils blue-black, chromophobes light blue
to colorless. Note the regional variations in the distribution of the various cell types. Note also
that in this pituitary there is a large colloid cyst (Rathke’s pouch).
Review the various hormones secreted by the basophils and acidophils (as defined in the
trichrome stains) of the pars distalis.
This preparation demonstrates the Herring bodies (large magenta-stained swellings on the
neurosecretory axons) in the neural lobe. What do Herring bodies represent? What hormones
might you expect to find in these structures?
ADRENAL GLAND
Each of the paired adrenal glands is in fact two glands. The outer mesodermally derived cortex
is composed of cells that secrete steroid hormones. The neural crest-derived cells of the
medulla are innervated by preganglionic fibers of the sympathetic nervous system and secrete
catecholamines.
This slide illustrates clearly the classical zonation of the adrenal gland. You should be able to
distinguish 5 zones in the organ: 1) the outer connective tissue capsule, 2) a thin zona
glomerulosa just beneath this, 3) the wide zona fasciculata, 4) a thin zona reticularis, 5) the
central medulla, within which lies the large central vein. Study the various zones in detail at
higher magnification.
1 2 3 4 5
63
Study this slide in the same way as the previous slide. The cortical zones are not as clear. The
tissue surrounding the central vein may not be medullary but instead may be in-growths of
cortical tissue. The chromaffin reaction following bichromate fixation results in differential
staining of epinephrine and norepinephrine cells, the latter are stained more darkly brown.
There are preganglionic sympathetic fibers arranged in nerve bundles in the medulla.
What hormone is produced by the zona glomerulosa? By the zona fasciculata? By the zona
reticularis? By the medulla? What hormones regulate the function of the cortex? How is
medullary function regulated?
THYROID
The thyroid gland consists of two populations of cells of different origin, histological
arrangement, and function. The follicular cells secrete thyroxine and triiodothyronine. These
hormones regulate development and metabolic rate. The parafollicular cells are of neural crest
origin and secrete calcitonin. This hormone is one of the factors regulating calcium and
phosphorous balance in the body.
64
PARATHYROID
The parathyroid glands are located on the posterior aspect of the thyroid. There may be
anywhere from 2 to 6 glands in an individual. Cells in these glands secrete parathyroid
hormone, which acts to increase calcium resorption from bone and in the renal tubules. It also
acts to increase the synthesis of the active form of Vitamin D. Vitamin D, in turn, increases the
absorption of Ca++ from the small intestine.
The endocrine component of the pancreas consists of multiple spherical groups of epithelial
cells embedded as nodules in the exocrine pancreas. The cells in the Islets of Langerhans are
not arranged into acini (as in the exocrine pancreas) but in irregular cords and clumps
surrounded by a rich capillary plexus. Note that the islets are not separated from the acinar
tissue by a capsule. The function of the islets is to control carbohydrate metabolism. The alpha
cells secrete glucagon, which raises blood sugar, and the beta cells secrete insulin, which
lowers it. There are several other biologically interesting peptides that are made by other cells
of the islets. The most important identified to date is somatostatin, which is made by the delta
cells. Its release locally inhibits both insulin and glucagon secretion. These cell types cannot
be differentiated by light microscopy.
Islets of Langerhans are clearly visible interspersed within the basophilic acini of the exocrine
pancreas. Keep in mind the classes of hormone producing cells are not distinguishable. For a
more detailed description of the exocrine pancreas see part two of the gastrointestinal system
lab on page 82.
ELECTRON MICROGRAPHS
QUESTIONS
Learning objectives:
1. Understand and identify the components of the male reproductive system and the
associated glands.
2. Understand and identify the stages in spermatogenesis and the cells that play essential
roles in this process.
3. Be aware of the importance of the blood-testis barrier.
The male reproductive system consists of the testes, the excretory ducts and associated
glands, the penis, and the scrotum. The testes contain many seminiferous tubules, which are
lined by a germinal epithelium consisting of germinal elements (spermatogonia,
spermatocytes, and spermatids) and Sertoli cells. Lying between the seminiferous tubules are
the interstitial cells of Leydig, which produce the male sex hormones (androgens). When
sperm leave the seminiferous tubules they pass through the following series of ducts:
Ducts Characteristics
1. tubuli recti short, straight tubules lined by cuboidal or columnar epithelium (Sertoli
cells); site of fluid secretion.
4. ductus
epididymis long coiled duct with thin smooth muscle coat and a pseudostratified
epithelium; columnar cells (called principal cells) which bear non-motile
processes known as stereocilia are interspersed with rounded basal
cells (found near the basement membrane); site of fluid absorption and
sperm storage and maturation.
5. ductus deferens thick muscular wall (to move spermatozoa toward ampulla) lined by
pseudostratified columnar epithelium with some stereocilia.
7. ejaculatory duct narrower than ampulla; runs through prostate gland to empty into
urethra.
The male sex accessory glands are the paired seminal vesicles, the prostate gland, and the
paired bulbourethral glands. The duct of each seminal vesicle unites with the ampulla of a
ductus deferens to form a common ejaculatory duct. The prostate gland surrounds the
ejaculatory duct and the prostatic urethra, and secretes into the latter.
At low magnification identify the tunica albuginea, the fibrous capsule surrounding the testis.
The mediastinum (not visible on this slide) is the mass of acidophilic connective tissue at one
67
pole through which the major vessels enter and leave the testis. The rete testis is located in
the mediastinum.
In the interstitium (between the seminiferous tubules) identify Leydig cells, which are large
eosinophilic cells. Why are they eosinophilic?
Note the similarity of the lining epithelium of the ductus deferens with that of the epididymis.
Characteristic of this organ is the stellate appearance of the lumen, and thick muscularis. The
muscularis consists of inner longitudinal, middle circular, and outer longitudinal layers.
Observe the arteries, veins and nerves that surround and penetrate the ductus deferens.
SEMINAL VESICLE
The highly folded lumen of the seminal vesicle appears as separate cavities when the organ is
sectioned. Note that there are no discrete secretory alveoli in the seminal vesicle; instead the
entire lining membrane of the saccular gland is thrown into a series of complex, high, thin
folds. The lining epithelium is generally simple columnar or pseudostratified, and basal cells
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are frequently seen, as in the epididymis and ductus deferens. The lamina propria contains
connective tissue and smooth muscle cells. The seminal vesicle is embryologically derived
from the ductus deferens, and like the latter, it has a prominent muscularis. This provides for
the expulsion of seminal vesicle fluid during ejaculation. The acidophilic secretory material in
the lumen of the gland is rich in fructose, thought to serve as an energy source for
spermatozoa following ejaculation. Contrary to the implications of its name, the seminal vesicle
is not a site of spermatozoa storage.
PROSTATE GLAND
Examine the Masson-stained section. At one surface, a prominent fold represents the cut
surface of the prostatic urethra. Also evident are the elongate tubules forming the parenchyma
of the gland and the dense fibrous connective tissue capsule. Locate the region of the
prostatic urethra and study its lining epithelium. Compare its transitional epithelium with the
epithelium lining the ducts and glands of the prostate, which can be cuboidal, columnar or
pseudostratified. The tubulo-alveolar glands of the prostate are embedded in an abundant
stroma of fibro-elastic connective tissue, which is interlaced with strands of smooth muscle.
Numerous concretions (corpora amylacea) occur in the lumen of the glands and ducts. These
tend to increase with age. Fixation is much better in the H & E sections, and it should be
studied for the structure of the lining epithelium of the glands.
The corpora cavernosa have been dissected away from the rest of the penis. Examine the
central penile urethra and the surrounding blood-filled vascular sinuses that comprise the
erectile tissue of the corpus spongiosum. Note that the lining epithelium of the penile urethra
has a stratified columnar or stratified cuboidal appearance. Study the erectile tissue
surrounding the urethra and observe that the trabeculae between blood sinuses contain
smooth muscle and connective tissue fibers. The connective tissue capsule surrounding the
corpus spongiosum is not as thick as that surrounding the corpora cavernosa.
ELECTRON MICROGRAPHS
QUESTIONS
Learning objectives:
1. Understand and identify the components of the female reproductive system.
2. Understand and recognize the stages of follicular development in the ovary. Be aware of
the hormonal function of the ovary.
3. Understand the effects of the hormonal environment on the endometrium.
4. Know the structure and function of the placenta.
5. Be able to relate the appearance of the mammary glands to their functional state.
OVARY
There are 3 sections of ovary on this slide. At low power note the general division of the ovary
into an outer cortex containing follicles in various stages of development and an inner medulla
containing numerous blood vessels and dense fibrous connective tissue.
Identify;
Lining epithelium (classically called “germinal epithelium”) - a simple cuboidal covering the
ovary, continuous with the mesothelium of the peritoneum.
Secondary (antral) follicles - 1o oocyte surrounded by granulosa cells among which fluid-filled
spaces are coalescing into a single space, or antrum. Outside the basal lamina of the
granulosa layer, the theca has differentiated into a theca interna and a theca externa.
Mature preovulatory (Graafian) follicle - characterized by a very large, central antrum. Zona
pellucida is very conspicuous. 1o oocyte is surrounded by a layer of granulosa cells (the
corona radiata) and rests on a small mound of granulosa cells called cumulus oophorus.
Atretic follicles - Note that follicles may undergo atresia during any stage of development.
Atresia is often first recognized in the granulosa cells as the nuclei become apoptotic and
there is a loosening of the cells.
Corpus luteum – Following ovulation follicular cells (both granulosa and luteal) fold into the
empty follicle and undergo luteinization. Following fertilization the corpus luteum becomes a
large, steroid-producing organ.
Corpus albicans – If fertilization does not occur, the corpus luteum regresses. The corpus
albicans is the connective tissue scar remaining from a degenerated corpus luteum.
70
unilaminar
primordial follicle multilaminar
primary follicle
primary follicle
secondary follicle
mature follicle
Note the scarcity of primary follicles, suggesting that this ovary is from an older woman.
Identify a corpus albicans (the connective tissue scar remaining from a degenerated corpus
luteum.
Compare the development of this corpus luteum of pregnancy (probably from the first
trimester) with that of the recently formed corpus luteum of slide #63. Note particularly the
increase in thickness of the granulosa luteal layer as compared to the thin, peripheral zone of
theca luteal cells. The extensive vacuolization of the granulosa luteal cells is due to the
extraction of its abundant lipid droplets. This reflects the importance of the corpus luteum
(particularly the granulosa lutein cells) as the primary ovarian source of the steroid hormone
progesterone.
What are the fine structural specializations of ovarian cells involved with the production of
steroid hormones?
Be certain that you understand the changes that occur within the follicle during follicular
development.
Consider the hormones of the anterior pituitary involved in follicular growth and ovulation.
There are sections taken from the ampulla of two uterine tubes: one in the middle of the
menstrual cycle and the other 2.5 months pregnant. Note the highly convoluted surface of the
mucosa. These folds decrease progressively from the ovarian (infundibular) end of the tube to
the uterine (isthmus) portion.
The muscularis becomes progressively thicker toward the uterine end of the tube. Ciliary
beating is the mechanism of movement of the ovum toward the uterus. Identify both ciliated
and secretory epithelial cells on the mucosal surface. The uterine tubes are a common site of
occlusion after pelvic inflammatory disease, resulting in sterility.
UTERUS
Identify the specialized uterine mucosa (endometrium) and the muscularis (myometrium). The
endometrium is characterized by its cyclic changes under the influence of ovarian hormones. It
is important to understand the interrelationships among the pituitary, ovary, and uterus during
different stages of the menstrual cycle.
The proliferative stage follows
menstruation and is characterized by
the repair of the endometrium and the
proliferation of relatively straight,
tubular uterine glands. Note the rather
dense, cellular appearance of the
endometrial stroma (region between
glands) at this stage. Coiled
endometrial arterioles are not readily
evident during the proliferative stage.
Left to right: spongy zone, stratum basale, myometrium
What is the primary ovarian hormone
stimulating the endometrium during this stage?
Secretory endometrium
72
CERVIX
What are some of the possible functions of cervical mucus? In what other regions of the body
does one observe an abrupt junction between simple columnar and stratified epithelia?
PLACENTA
The placenta may be defined as an apposition or fusion of the fetal membranes with the
uterine mucosa for the purpose of physiological exchange. The period of placentation is
initiated by the attachment of the blastocyst to the endometrium, and it is terminated by the
delivery of the newborn infant at the time of parturition. The placenta is the first organ to be
differentiated, and performs functions analogous to those of the lung (gas exchange), intestine
(nutrient absorption), kidney (excretion and ion regulation), liver (synthesis of serum proteins,
steroid metabolism), pituitary (synthesis of hormones including gonadotropic and prolactin-like
hormones), and gonads (incomplete synthesis of progestins and estrogens).
Only the fetal surface of the placenta is present on this slide, so that the attachment of the fetal
villi to the uterus cannot be studied. The fetal portion of the placenta consists of the chorionic
plate, composed of an outer layer of trophoblast and an inner layer of vascularized extra-
embryonic mesodermal connective tissue. The bulk of the placenta fetalis consists of
outgrowths of villi from the surface of the chorionic plate. The villi are sectioned in many
73
different planes, and their attachment to the chorionic plate may not be evident. Attached to
the inner (fetal) surface of the chorionic plate is the amnion, consisting of an inner squamous
amniotic epithelium and an outer layer of avascular mesoderm.
Study the chorionic villi in detail, and identify all of the layers that separate the maternal and
fetal blood. These are:
Gases, nutrients, metabolites and other substances must pass through these layers to move
from one circulation to the other. In life maternal blood fills the intervillous space, but it is
generally washed out during tissue preparation.
Mitotic figures are occasionally seen in the cytotrophoblast, but not in the syncytiotrophoblast.
Note the loose appearance of the cells forming the cores of the villi, and compare this with the
condition in the villi at 6 months gestational age. Occasional nucleated fetal red blood cells,
characteristic of earlier stages, can still be observed in the fetal vessels of the villi.
Be certain that you know the layers that form the separation
between fetal and maternal blood in the placenta.
BREAST
The mammary gland is a cyclic organ, varying in size and structure in response to hormones
from the adrenal, ovary and pituitary. The two slides in your collection represent two of the
extremes normally encountered. You should realize that this gland is normally subject to a
great deal of variation.
The breast contains a duct system, lobes, and lobules. This network
of ducts begins at the nipple with the excretory lactiferous duct,
which branches as it extends into the collagen and adipose tissue of
the breast until it eventually branches into terminal duct lobular units.
The terminal duct lobular unit consists of interlobular stroma,
interlobular duct, terminal duct and acini, and surrounding fat.
Identify these units on low power. With higher power, note that the
ducts and acini are lined by simple cuboidal or columnar epithelium
and surrounding myoepithelial cells.
The mammary gland in the prepubertal female and male has a similar appearance. There is
abundant connective tissue with embedded lactiferous ducts, ending in minimal lobule
formation
Unsaturated lipid in the apical cytoplasm of the alveolar cells and in the milk in the lumina is
stained black by reduced osmium tetroxide. Because osmium penetrates very poorly the
tissue is well stained only at the periphery of the section. The gland is separated into lobules
by dense connective tissue that is continuous with the dermis. The connective tissue
surrounding the alveolus is much more delicate (although compressed here) and is continuous
with the papillary layer of the dermis. The secretory alveoli are very well supplied with
capillaries.
What are the major hormones that are responsible for the cyclic changes in the mammary
gland?
ELECTRON MICROGRAPHS
GASTROINTESTINAL SYSTEM I
Learning objectives:
1. Know how the fundamental organization of the GI tract is modified along its course from
the esophagus through large intestine.
2. Know the cell types in the mucosa of the GI tract and their function.
The digestive system consists of the oral cavity, the pharynx, the alimentary tract (canal), and
the anal canal. There are both intrinsic and extrinsic glands, which may secrete digestive
enzymes or mucus to facilitate the digestion and transport of ingested food. The intrinsic
glands lie within the mucosa or submucosa of their organ of origin. The extrinsic glands
communicate with their organ of origin through ducts. The extrinsic digestive glands are the
major salivary glands including the parotid, sublingual and submandibular (submaxillary)
glands; the pancreas; and the liver. These glands will be discussed in the following lab.
Organs of the digestive tract typically have 4 concentric coats. Proceeding outward from the
lumen these are: (1) the mucosa (mucous membrane), (2) the submucosa, (3) the muscularis
(muscularis externa), and (4) the adventitia or serosa. (Refer to diagram below).
1. The mucosa has three components: (a) the epithelium and its underlying basement
membrane, (b) a thin underlying layer of loose, cellular connective tissue, the lamina
propria, and (c) a relatively thin layer of smooth muscle called the muscularis mucosae.
The latter may consist of both circular and longitudinally arranged layers.
2. The submucosa is composed of a layer of dense, irregularly arranged connective tissue
that contains nervous tissue (the submucosal plexus of Meissner) as well as blood vessels.
3. The muscularis externa consists of at least 2 layers of smooth muscle, an inner circular
and outer longitudinal layer. Connective tissue separating the muscle layers contains
nerves (myenteric plexus of Auerbach) and blood vessels.
4. The outermost layer or adventitia consists of a thin layer of loose connective tissue. Where
the digestive system is covered by peritoneum the adventitial layer is called the serosa.
77
ORAL CAVITY
The epithelial lining of the oral cavity is of the stratified squamous type. In contrast to the skin it
is non-keratinized. The major salivary glands arise as invaginations of the oral epithelium
during the second month of embryonic development, and they are involved with the secretion
of the watery, mucus, and enzymatic content of saliva. A description of these glands can be
found in the following lab.
TONGUE
The tongue is easily recognized because of its interlacing bundles of skeletal muscle that are
disposed in three plane, and by its covering of stratified squamous epithelium that is elevated
on the dorsal surface of the tongue into papillae. There are three types of papillae in humans,
filiform, fungiform, and circumvallate.
As in both the oral cavity and in the pharynx, the mucosal surface of the esophagus is lined by
stratified squamous epithelium that is non-keratinized in humans. In herbivores, the
esophagus has a keratinized epithelium. The well-developed muscularis externa and the
stratified squamous epithelial lining are well adapted for the rapid transport of food from the
pharynx to the stomach. Diagnostic features of the esophagus are the combination of stratified
squamous surface epithelium and the considerable thickness of the muscularis mucosae (up
to 0.2 - 0.4 mm thick). The upper third of the muscularis externa contains mostly skeletal
muscle, the middle third contains a mixture of skeletal and smooth, and the lower third
contains only smooth muscle.
78
Examine the wall of the esophagus starting with the stratified squamous non-keratinized
epithelium. Underlying the epithelium is a layer of loose connective tissue and the muscularis
mucosae. Note the intermingling of both skeletal and smooth muscle in the muscularis
externa. Use this slide to review the histology of smooth and skeletal muscle, comparing them
with the adjacent connective tissue of both the lamina propria and the submucosa. Also,
identify the various types of vessels within the submucosa, as well as other layers.
STOMACH
The stomach extends from the esophagus to the duodenum. It is divisible into the cardiac,
fundus, body, and pyloric regions. The distensible stomach is involved in both the mechanical
and chemical breakdown of food, and also serves as a temporary reservoir. Its simple
columnar epithelium is specialized for secretion. The gastric mucosa contains gastric pits
(foveolae), which are surface invaginations that also serve as the ducts of the underlying
intrinsic gastric glands. Three basic cell types contribute to the secretion of gastric juice, and
each has a characteristic appearance under the light and electron microscope. All of these cell
types can be seen in the fundus and body of the stomach.
fundus
esophagus
cardia
body
pyloric
sphincter
pylorus
Stomach
duodenum
Identify the mucosa, submucosa, and muscularis externa. Locate the following elements of the
mucosa: the luminal surface mucous secreting cells, the gastric pits and the cells lining them.
Parietal cells are particularly prominent, and chief cells and mucous neck cells are present.
Note the loose connective tissue surrounding the gastric pits, the muscularis mucosae, which
forms a boundary between the mucosa and
submucosa, and the blood vessels in the submucosa.
The cell bodies and nerve fibers of the Meissner’s
plexus are found in the submucosa. These are not
clearly visible on this slide. Examine the muscularis
externa and notice that the smooth muscle is oriented
in several different planes. A serosa covers the
external surface of the gland in this section. The
myenteric plexus (Auerbach's plexus) is located
between the external and adjacent inner layers of Auerbach’s plexus
smooth muscle and is clearly evident on this slide.
SMALL INTESTINE
epithelium that lines the pits or villi. In the stomach the cells all have a uniform appearance,
since they are all mucus-secreting cells. In the intestinal villi however, most of the cells are
absorptive cells, and interspersed between these are the characteristic mucous-secreting
goblet cells. Goblet cells of the intestine will stand out when the slide is scanned under low
power. In addition, a brush border can sometimes be seen on the free surface of the
absorptive cells in well-preserved intestinal villi. To what ultrastructural feature does the brush
border correspond? The duodenum is also characterized by the presence of mucous-secreting
duodenal glands (of Brunner) in its submucosa.
Identify the three components of the mucosa: epithelium, lamina propria and muscularis
mucosae. Circular folds called plicae circulares are visible at low power on this slide. They
further increase surface area for absorption. Note the submucosal glands of Bruner. The two
layers of the muscularis externa are present and outside these is the adventitia. Between the
two layers of the muscularis externa, identify elements of the myenteric plexus. The pancreas
can be seen beneath the muscularis externa. This will be studied in the next lab.
Identify the components of the wall of the small intestine. Note the
presence of diffuse lymphatic tissue in the mucosa (GALT). In the
ileum there are accumulations of lymph nodules called Peyer’s
patches. At the base of the intestinal crypts Paneth cells are found.
These are characterized by the accumulation of large acidophilic
granules in their apical cytoplasm, and by their strongly basophilic
basal cytoplasm. The appearance of these cells is characteristic of
enzyme-secreting cells. These cells secrete the anti-bacterial enzyme
lysozyme.
LARGE INTESTINE
Note the regularity of the intestinal mucosa crypts and the lack of villi. Also note the
abundance of goblet cells. These features are diagnostic. This is a good slide in which to
review the structure of arteries, veins, and the peripheral autonomic plexus. Enteroendocrine
cells are present at the base of the crypts, but are difficult to identify. These cells secrete
hormones through the basal cell surface into the capillaries.
ELECTRON MICROGRAPHS
GASTROINTESTINAL SYSTEM II
Learning objectives:
1. Understand the structure and function of the salivary glands.
2. Understand the structure and function of the liver, including blood and bile flow. Be sure to
know the products of the hepatocytes.
3. Understand the structure of the gall bladder and bile duct.
4. Understand the structure and function of the exocrine and endocrine pancreas.
The major salivary glands (as well as the pancreas) are examples of exocrine glands, meaning
the products of secretory cells are delivered via ducts to their functional site. These glands are
classified according to: (1) the organization of the cells in the secretory portion of the gland
e.g. tubular, alveolar (synonymous with acinar) or tubulo-alveolar (both types are present), and
(2) the configuration of the cells that form the excretory duct (or ducts). If the duct is
unbranched, the gland is called a simple gland. If however, the duct branches the gland is
known as a compound gland.
This gland is purely serous in the adult human, so that all of the secretory units or acini have a
similar appearance. A fibrous connective tissue capsule surrounds the gland and sends septa
inward that subdivide the gland into lobules.
Scan the slide on low power and observe that within each lobule
there are several prominent ducts with a more distinct lumen, which
stand out sharply from the surrounding acini because of their
acidophilia. These are the intralobular or striated ducts. Larger
interlobular or excretory ducts are embedded in the extensive
interlobular connective tissue, and their epithelium may be either
simple columnar, pseudostratified, or stratified. Goblet cells are
occasionally seen in the interlobular ducts. The secretory acinus is
connected to the intralobular duct by a thin intercalated duct, which
is difficult to see in this slide. These intercalated ducts consist of a
cuboidal epithelium, and their diameter is less than that of either
the acinus or the intralobular duct.
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LIVER
The liver is the largest gland in the body, and it has both an endocrine and exocrine function.
The liver is organized into lobes surrounded by a thick connective tissue capsule. Each lobe is
subdivided into lobules by looser connective tissue (Glisson's capsule). In the human, this
connective tissue does not completely outline the lobule. It can be seen best in regions where
there are sections of the bile duct and the hepatic artery. The structural plan of the liver is a
reflection of its vascular supply. Blood enters the liver via the hepatic artery and portal vein,
which send branches to the hepatic lobules. Within the lobules, blood travels between the
plates of hepatic cells in sinusoids toward a central vein.
It leaves the lobules via branches of the hepatic vein.
Hepatic triad
84
In addition to recognizing the landmarks of the classic lobule, be aware of the boundaries of
the portal lobule and liver acinus.
Identify the vessels and structures discussed above. Notice that a thin space is present
between the endothelial cells lining the sinusoids and the parenchymal cells. This is the space
of Disse, and it is in continuity with the lumen of the sinusoids via small spaces between the
endothelial cells that form the wall of the sinusoids.
This slide should be examined as above. In addition, the bile canaliculi are revealed as
delicate tubules that course between the apposed surfaces of the parenchymal cells. These
are seen best in regions where the plates of cells are two cells thick.
GALL BLADDER
Islet of Langerhans
surrounded by serous glands
85
delivered through a duct system that is similar to that in the salivary glands: intercalated duct
to intralobular duct to interlobular duct. A diagnostic feature of the exocrine pancreas is the
presence of centro-acinar cells. These cells form the initial portion of the intercalated duct. The
pale-staining nuclei of the centro-acinar cells appear in the center of an acinus (hence their
name).
For a more detailed description of the endocrine portion of the pancreas see the endocrine
glands lab on page 61.
In the exocrine portion of the pancreas on this slide note that the basal cytoplasm of the acinar
cells is highly basophilic. Zymogen granules at the apex are very acidophilic. The cytoplasm of
centro-acinar cells and duct cells is relatively unstained. This slide clearly demonstrates the
duct system in the pancreas. Islets of Langerhans are clearly visible, however the classes of
hormone producing cells are not distinguishable.
ELECTRON MICROGRAPHS
QUESTIONS
1. Why can the liver be characterized as both an exocrine and endocrine organ?
ANSWERS TO QUESTIONS
What subcellular organelle is responsible for attracting the basic stain? Rough endoplasmic
reticulum.
Why are the microvilli not visible on all cells lining the lumen? Depending on the orientation of
the section, certain cellular components may not be visible in all cells.
QUESTIONS
1. What are serial sections and why are they important? A continuous series of sections,
which reveal structures in three dimensions. Serial sections are important for
visualizing the three dimensional structure of the tissue in order to differentiate artifact
from pathology.
2. What is the relationship between heterochromatin and the synthetic activity of DNA?
Heterochromatin is transcriptionally inactive DNA. A euchromatic nucleus is relatively
unstained because the genetic material is being read.
6. How many membranes comprise the nuclear envelope? Two. The wall of a
mitochondrion? Two.
7. What are the cytological and functional differences between cilia and microvilli? Cilia
are motile microtubule based structures; microvilli are non-motile actin based
structures.
2. EPITHELIUM
Why do the nuclei appear at different levels in tangential sections? Depending on the
orientation of the tissue during sectioning, the orientation of the cells on the slide can appear
different than the orientation of the cells in tissue.
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What is the basis for the PAS stain? Most carbohydrates react with periodic acid to produce
aldehydes, which convert the colorless Schiff reagent to pink, or magenta.
What types of intercellular junctions are commonly found in epithelia? Junctional complexes
(tight junctions plus zonula adherens), desmosomes, gap junctions
#5 Trachea
Identify the two major types of cell that reach the lumen. What are their characteristics? Simple
columnar ciliated epithelial cells and goblet cells (appear empty). What is responsible for the
eosinophilic line at the apex of the majority of the cells? Basal bodies
Which part of the slide image corresponds to the esophageal lumen? The bottom of the image
would correspond to lumen of the esophagus because the epithelium is oriented facing the
bottom of the image.
3. CONNECTIVE TISSUES
Where is the majority of brown fat found in humans? In newborns on the upper back,
functioning in temperature regulation.
QUESTIONS
1. Are reticular fibers distinguishable in tissue stained with H&E? No
Where else does elastic cartilage occur in the body? Eustachian tube and epiglottis.
QUESTIONS
1. What are the mechanisms of cartilage growth? Appositional and interstitial growth
2. What is the distribution of blood vessels in cartilage, and how does this relate to the
nutrition of cartilage? Blood vessels are found only in the perichondrium. Nutrition by diffusion
through ground substance
The cementing lines that delimit the Haversian systems may appear refractile or slightly
basophilic. What accounts for this basophilia? Proteoglycans.
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QUESTIONS
1. What structures are found within Haversian canals? Capillaries and nerves
2. Is the osseous lamella adjacent to the Haversian canal the youngest or the oldest lamella of
a particular osteon? The youngest
Be sure you know how cartilage and bone differ morphologically, functionally, and with respect
to blood supply.
Cartilage Bone
What structure in mature bone is created by the zone of resportion? The marrow canal
QUESTIONS
5. NERVE
How is the structure of the dorsal side of the spinal cord different from the ventral side? The
ventral horns are more rounded. The cell bodies in the ventral horn are larger because they
are involved in motor function. In this slide ventral is at the bottom of the image.
To what structures at the electron microscopic level do the Nissl bodies correspond? RER,
free ribosomes
What are the three layers of meninges? Dura mater (outermost), Arachnoid matter, Pia mater
(innermost). Is there anything inside the central canal? Cerebrospinal fluid
What is the functional consequence of the location of these neurons (that is, the
parasympathetic ganglion) near the target organ? Nerve impulses reach the target organ more
quickly via the parasympathetics than sympathetics. Impulses move more slowly along
unmyelinated axons, and the unmyelinated postganglionic axons are much shorter in the
parasympathetic system than the sympathetic system.
Locate neuronal cell bodies within the ganglia. What other types of cell would you expect to
find in these ganglia? Glia
Find some nodes of Ranvier. What is their role? Site where depolarization occurs in
myelinated nerves
What are the cells within the nerve whose nuclei are stained? Schwann cells, fibroblasts
6. MUSCLE
Define a sarcomere. Be sure you know what the electron microscope has revealed about its
fine structure. Know the structural changes that occur in a sarcomere during contraction and
the theory that has evolved from electron microscopic studies to explain muscle contraction. A
sarcomere is the basic contractile unit of a muscle cell, repeating sarcomeres comprise a
myofibril. The Z-bands are considered the ends of a single sarcomere, with the H-band in the
center. As the muscle contracts, the Z-bands move closer together and the I-band and H-band
shorten in length as the actin thin filaments are moved along the myosin thick filaments. The A
band remains the same length because the length of the myosin unit does not change.
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What is the position of the nuclei? Central. Do the myofibrils pass through intercalated discs?
No. How can you distinguish cross sections of cardiac muscle fibers from those of skeletal
muscle fibers? Central nuclei, intercalated discs, branching fibers
Note that when the muscle cells are cut in cross section, there are interruptions in the basal
laminae. What is responsible for these discontinuities? Gap junctions
QUESTIONS
1. Why do smooth muscle fibers in cross section have different diameters and why do some
of these fail to show nuclei? Smooth muscle cells have tapered ends. Since the cells
interdigitate different diameters would be revealed in a particular plane of section and the
plane of section does not always go through the nucleus.
Be sure you know the biochemical composition of the cytoplasmic granules of neutrophils
(polymorphonuclear leukocytes), eosinophils, and basophils.
QUESTIONS
1. What is the functional significance of the cytoplasmic staining affinities of the basophilic
erythroblast, polychromatophilic erythroblast, normoblast and erythrocyte?
Basophilic erythroblast - ribosomes predominate for production of hemoglobin and
transferrin receptors.
Polychromatophilic erythroblast - hemoglobin synthesis beginning.
Normoblast - hemoglobin fills cytoplasm, pyknotic nucleus.
Erythrocyte - anuclear cell, hemoglobin fills cytoplasm.
8. LYMPHOID TISSUES
Aggregates of lymphocytes are most common in the small intestine (Peyer's patches) and in
the vermiform appendix. Are these aggregates encapsulated or unencapsulated?
unencapsulated
QUESTIONS
1. Which lymphatic organs have afferent lymphatic vessels? Lymph nodes
3. What are the components of the blood thymic barrier? a perivascular connective tissue
sheath containing macrophages, a basal lamina, and an epithelioreticular cell sheath
4. Which of the lymphoid organs filters blood rather than lymph? Spleen
9. CARDIOVASCULAR SYSTEM
Is the myocardium thicker in the atrium or ventricle? Why? The myocardium is thicker in the
ventricle. The ventricle must create more force when contracting to deliver blood to the lungs
(right ventricle) or the entire body (left ventricle), whereas the atrium only has to deliver blood
to the ventricle.
QUESTIONS
1. What are the three layers of the heart? Endocardium (inner), myocardium (middle,
comprised of cardiac muscle), epicardium (outer).
2. What types of capillaries are depicted on the covers of this manual and where might such
capillaries be found? Front cover (fenestrated: endocrines, mucosa of gut, gall bladder)
and inside the back cover (continuous: muscle, brain, lung)
10. SKIN
What criteria do you use to distinguish between the esophagus and the trachea? Different
types of epithelium, absence of cartilage rings in esophagus
QUESTIONS
1. What is the distinctive structural component of the wall of the trachea? Cartilage rings
2. What does the EM demonstrate regarding the air-blood barrier? Capillaries are interposed
between epithelial lining cells of adjacent alveoli
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3. What are the features that distinguish bronchi from bronchioles? See chart at beginning of
lab description.
4. What is the importance of elastin in the respiratory system? Allows for expandability and
return to original volume during expiration.
5. Which cells are responsible from keeping the lungs free from obstructing particulate
matter? Macrophages (dust cells) How do they carry out this function? Phagocytosis
What is the functional significance of the occurrence of a brush border in the proximal tubule?
The brush border increases the surface area, facilitating the reabsorption that occurs in the
proximal tubule
What is the functional significance of the occurrence of a brush border in the proximal tubule?
Expanded apical surface maximizes reabsorption
What are the components of the arterial portal system of the kidney? Afferent arteriole,
glomerular capillaries, efferent arteriole
What are the components of the blood urinary barrier in the glomerulus? endothelial cell
cytoplasm (fenestrations); three-part glomerular basement membrane composed of type IV
collagen and polyanionic glycosaminoglycans; slit-diaphragms extending from the podocytes.
What kinds of cells are in the neurohypophysis? Pituicytes (glia), endothelial cells
QUESTIONS
1. What is the functional significance of the hypothalamo-hypophyseal system? It allows for
rapid and direct delivery of hypothalamic products with releasing and inhibiting effects on
anterior pituitary cells
2. What factors control the secretion of parathyroid hormone? Serum calcium levels
3. What is the consequence of parathyroid hypofunction? Lowered blood calcium level can be
life threatening.
4. How does parathyroid hormone act? Raises serum calcium levels by acting on osteoblasts
that in turn activate osteoclasts (release calcium from bone).
Why are the Leydig cells eosinophilic? They contain numerous mitochondria.
QUESTIONS
1. What is the difference between spermatogenesis and spermiogenesis?
Spermatogenesis is entire the process of formation of sperm from stem cell to
spermatozoan. Spermiogenesis is the maturation process from spermatid to
spermatozoan.
2. What is the effect of castration on the accessory sex glands (prostate and seminal
vesicles)? Atrophy.
3. Where is the primary source of testosterone? The Leydig cells.
4. Where is the principal site of storage and mobility acquisition of spermatozoa in the male
reproductive system? Epididymis.
5. Follow the passage of spermatozoa from the seminiferous tubules of the testis up to
ejaculation. Seminiferous tubules, tubuli recti, rete testis, ductuli efferentes, epididymis,
ductus deferens, urethra.
6. Which organs are the major sources of seminal fluid? Seminal vesicle, prostate gland,
bulbo-urethral.
7. What are the components of the blood-testis barrier and what is its significance? Tight
junctions between Sertoli cells isolate developing sperm from the vasculature (prevent their
immunological rejection).
What are the fine structural specializations of ovarian cells involved with the production of
steroid hormones? Mitochondria with tubulo-vesicular cristae, SER, lipid droplets.
What is the primary ovarian hormone stimulating the endometrium during this stage?
Estrogen.
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What is the dominant ovarian structure during the secretory stage? Corpus luteum.
Which ovarian hormone is necessary for the maintenance of the secretory stage of the
endometrium? Progesterone.
Which zones of the endometrium may be lost during menstruation? Functionalis (compactum
and spongiosum).
What are some of the possible functions of cervical mucus? Impedes sperm entry except in
peri-ovulatory period when the mucus in less viscous
In what other regions of the body does one observe an abrupt junction between simple
columnar and stratified epithelia? Gastro-esophageal junction, colo-rectal junction.
PLACENTA
BREAST
What are the major hormones responsible for the cyclic changes in the mammary gland?
Estrogen and progesterone.
How can you diagnose whether you are looking at the upper or lower portion of the
esophagus? By the presence and/or proportion of skeletal muscle.
To what ultrastructural feature does the striated border correspond? Microvillus border.
In the lamina propria there are macrophages with irregular-sized PAS-positive inclusions.
There are also mast cells. What accounts for the intense PAS-positivity of these cells?
Carbohydrate groups
Bile is released under stimulation from the small intestine. What hormone is responsible for
this? CCK.
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QUESTIONS
1. Why can the liver be characterized as both an exocrine and endocrine organ? Secretion of
bile into ductules, release of products into sinusoids (bloodstream)
2. What are the secretory products of the exocrine pancreas? Digestive enzymes:
trypsinogen, lipase, amylase, etc., in inactive state
3. What is the major factor controlling insulin secretion? blood glucose levels.
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HISTOLOGICAL TECHNIQUES
In order to study tissues with a microscope they must be preserved (fixed) and cut into
sections thin enough to be translucent. The process of fixation is briefly described in the next
section. Fundamentally it consists of a chemical or physical method of killing the tissue and
yet retaining characteristic peculiarities of shape and structure. Following fixation, blocks of
tissue must be cut into thin sections. One way is to make a firm block by freezing fresh or
fixed tissue. Other techniques involve dehydration in alcohols and infiltration with paraffin, or
some similar agent - a process called embedding. Sections 3 to 10 microns (3 to 10
thousandths of a millimeter) in thickness are cut on steel knives mounted in an instrument
called a microtome, which has a precise mechanical advance.
For electron microscopy the sections are considerably less than one ten-thousandth of a
millimeter (0.1 micron, µm) thick. This is accomplished by embedding the tissue in a plastic
such as Epon or araldite (epoxy resins) and cutting on special ultramicrotomes equipped with
a fine mechanical or thermal advance. Sections are cut with glass or diamond knives and
mounted on copper mesh grids.
In some cases, serial sections are required. For this technique, multiple consecutive sections
are prepared as slides. Using serial sections allows the 3D structure of the tissue to be
visualized. This is especially important in determining whether an abnormality is an artifact of
preparation or a pathologic process.
In work with the light microscope, it is difficult to recognize the various components of cells and
tissues without differential staining. The stains may react chemically or physically and a wide
variation is possible. The staining method can be altered to suit the needs of the examiner in
order to accentuate certain tissues or organelles.
Finally, in order to preserve the section which has been made from a block of fixed tissues and
stained, it is mounted on a glass slide and covered with a thin cover glass by means of a
transparent substance which hardens and seals the preparation to make it permanent. Some
tissues are stained and then mounted. More often the tissue is placed on the slide first and
then stained. The mounting medium used to attach the coverslip must have a refractive index
similar to that of the glass slide and cover slip to prevent distortion.
FIXATION
This process has two phases: 1) the coagulation or precipitation of the various components of
the tissues and cells and 2) their preservation in a state as nearly as possible like the living
condition by forming stable chemical compounds. The first phase carries with it an intrinsic
source of difficulty and error. The precipitation may be uneven and cause deposits to form
where no structure existed in the living cell. These are called "fixation artifacts". The second
phase also carries a source of difficulty because the compounds formed by some fixatives will
not take up some stains. It has not been possible to find an ideal fixative that 1) penetrates
quickly, 2) renders all parts of all cells permanent and 3) allows the use of all kinds of stains.
The reason for this is not difficult to understand. The cell is a highly complex mixture of
proteins, carbohydrates and fats. The ideal fixative would not only have to form stable
compounds with all of these, but also render them insoluble both in fat solvents and in water.
Some fixatives not only fail to preserve certain parts of the cell but actually dissolve or destroy
them. For example, acetic acid destroys mitochondria. Moreover, some fixatives change the
shape and relationship of parts of a tissue by shrinkage.
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Formalin
This is a good general fixative. Its effect is to cross-link membrane proteins by forming
covalent bonds. It is made by dilution of commercial formaldehyde (which is a 40% solution of
formaldehyde gas in water) in an aqueous phosphate buffer. The usual strength is 10% (or
4% of the gas). It penetrates rapidly, causes little distortion, does not destroy any of the
cellular constituents and can be followed by almost all staining methods. It hardens the
tissues very slowly, however, and does not protect them from the shrinking agents employed
in embedding and sectioning. For this reason it is often combined with other fixing agents.
Osmium
Osmium tetroxide (OsO4) preserves the cell in a form closer to the living than any other
fixative. Its great disadvantage is that it penetrates poorly and cannot be followed by many
stains. It is also used as a stain because it blackens fat and various lipid-containing materials
such as the myelin sheaths of nerve fibers, and makes them insoluble both in water and in fat
solvents. Osmium tetroxide solution, in various buffers, is a standard fixative for electron
microscopy.
EMBEDDING
Paraffin Embedding
Since water and paraffin do not mix, the first step in embedding with paraffin is to replace the
water in the tissues with a solvent that is miscible with paraffin.
Dehydration - is the first part of the process. It is usually accomplished by transferring the
block of tissue through a series of alcohol-water solutions beginning with 50 percent and
running up to water-free or absolute alcohol.
Clearing - The alcohol is replaced by Histoclear (a non-toxic substitute for xylol) or cedar oil,
which is readily soluble in alcohol, and in turn, is replaced by melted paraffin.
Embedding - The actual embedding takes place when the paraffin- infiltrated tissue is placed
in fresh paraffin and the latter allowed to cool. It is important to remember that the xylol and
other solvents will dissolve the fats of the tissues unless they are fixed by some special
chemical such as osmic acid.
Celloidin Embedding - Celloidin is dissolved in equal parts of absolute alcohol and ether. The
tissue is dehydrated in alcohol in the same way as for paraffin except that it is transferred from
absolute alcohol to a dilute solution of celloidin. As the alcohol and ether evaporate, they are
replaced by more concentrated celloidin. It is finally hardened in chloroform and stored in 80
percent alcohol. It is a much longer process than paraffin but causes much less shrinkage and
distortion. It is used especially in examination of the eye and brain.
Epoxy Embedding - Introduction of epoxy embedding media has greatly reduced artifacts due
to shrinkage and also has allowed thinner sectioning than was possible with paraffin. The
thinner sections (approximately 1 u) may be viewed after staining with the light microscope or
may be sectioned thinner and examined by electron microscopy.
STAINING
Stains react in two general ways: 1) They combined directly with the tissue, or 2) they require
that the tissues be treated first with an anchoring substance or mordant. Very few stains can
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be relied upon to color with the desired selectivity or intensity unless carefully controlled. This
may be accomplished by stopping at the desired intensity or removing excess with another
reagent.
Selective stains have been found for many of the different parts of the cell and for
characteristic elements in the tissues. Much of the selective action is due to the fixation and
previous treatment as well as to the subsequent staining and differentiation.
Impregnation is not really a staining process but it is considered as one of the staining
methods. The tissues are first placed in a solution of the salt of a heavy metal. The metal is
precipitated as a black deposit about certain structures. These stains are especially used for
study of neurons and glia of the central nervous system.
Basic dyes are cationic. They form salts with tissue anions (components that carry a net
negative charge), especially the phosphate groups of nucleic acids and the sulfate groups of
the glycosaminoglycans.
Basophilic is the term used to designate the components of a cell or tissue, which take up the
basic stain rather than the acid stain of a combination. Nuclei are basophilic.
Acid dyes are anionic. They form salts with cationic groups in cells and tissues, particularly
the ionized amino groups of proteins.
Acidophilic or oxyphilic is applied to parts, which show a greater affinity for acid dyes. The
cytoplasm is usually acidophilic. Eosinophilic components are cationic compounds that have
an affinity for that acid dye.
Mordants
A mordanting substance is considered part of the stain, and in this way it may change the
reaction of the stain. For example, hematoxylin is an acid, but as it is almost always used in
conjunction with alum or iron (the mordant) it becomes a basic stain.
Amphophilic is a term used to indicate that the tissue stains with both the basic and the acidic
dyes.
Neutrophilic. No special affinity for either the basic or acidic components of a dye.
Metachromasia refers to the production of a color during staining which is different from the
original color of the staining solution. Mast cell granules will stain a reddish-purple with
toluidine blue. Metachromasia is pH dependent. Many substances are only metachromatic
when stained as frozen sections. Usually they must be viewed immediately, if not sooner.
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THE FOLLOWING LIST INCLUDES THE STAINING METHODS USED ON THE SLIDES IN
THE LOAN COLLECTION. IT GIVES A BRIEF SKETCH OF THEIR SELECTIVITY, MODE
OF ACTION, AND PROCEDURE. THEY ARE ARRANGED IN ALPHABETICAL ORDER FOR
READY REFERENCE. THE ABBREVIATIONS ARE THOSE USED IN THE CATALOGUE OF
THE LOAN COLLECTION. YOU ARE NOT RESPONSIBLE FOR THIS MATERIAL.
Acid phosphatase reaction: This histochemical technique is used to recognize lysosomes due
to their acid phosphatase content. Sections are incubated in a solution containing a lead
phosphate. The phosphate is released by enzymatic activity of acid phosphatase (lysosomal
enzyme) and is precipitated as lead phosphate, and is then converted to lead sulfide a black
deposit.
Alkaline phosphatase (Al. P): The histochemical technique used for demonstrating the
enzyme, alkaline phosphatase, blackens the cells and tissue containing the enzyme. In
general, the degree of blackness is correlated with the quantity of enzyme present. Exact
localization is complicated by the fact that the enzyme may shift its intracellular position during
the histological procedure. Sections are incubated in a solution consisting of sodium
glycerophosphate and calcium nitrate. Through the action of the phosphatase, calcium
phosphate is precipitated in those regions where the enzyme is present. For visualization in
sections, the calcium phosphate is converted into cobalt phosphate and finally into cobalt
sulfide, which is black.
Azure II - Eosin (Az. II. E.): Nuclei are blue or purple. Basophilic material blue. Acidophilic
material red. Red blood corpuscles orange.
Berlin Blue (Prussian Blue): An insoluble particulate iron-cyanide compound, which is used for
the injection of blood and lymph vessels.
Best's Carmine: A specific stain for glycogen by which the glycogen granules are stained red.
The PAS (periodic acid Schiff reaction) also colors glycogen red and is more commonly used.
Bielschowsky's Silver Method: The reticular connective tissue fibrils are black. All other
structures, yellow or brown. An impregnation method, which depends on the reduction by
formalin of the easily reducible silver salt, silver ammonium hydroxide.
Bodian Silver method: Metallic silver is precipitated by the action of a reducing agent (either
exogenous or endogenous). The exogenous agent results in deposits on reticular fibers and
portions of the junctional complex (argyrophilia). An endogenous agent results in precipitation
on granules of enteroendocrine cells (the argentaffin reaction).
Cajal's Silver Stain (Cajal): Neurofibrils, axons and dendrites black. Other parts brown. The
general principle of the Cajal methods (and there are many modification) is the application of
photographic developers to tissues, which have been treated with silver nitrate.
Chrome Hematoxylin and Phloxin: The use of these dyes for the differential staining of the
alpha and beta cells of the islets of Langerhans was described by Gomori, 1941 (Am. J. Path.,
Vol. 17). The granules of the beta cells are stained a deep blue; those of the alpha are pink or
red. The D cells are not differentially colored.
Eosin (E): An acid dye. Colors cytoplasm red; red blood cells, orange. Used as a counter-
stain. See under H&E.
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Foots Silver: This is a modification of Bielschowsky's silver method. The thin collagen or
reticular fibers stain black, other tissues remain pale. Azocarmine is frequently used as a
counter-stain to color the cells and collagenous tissue red.
Giemsa: Methylene blue eosinate, azure B eosinate, azure A eosinate, methylene blue
chloride in methanol.
Golgi Silver Method (Golgi): A black deposit of reduced silver is laid down on the surfaces of
nerve cells and neuroglia cells so that the form of the cell body and its processes stand out
prominently in an almost colorless background. Only single cells here and there are selected
by the stain. The method consists essentially of immersing fresh pieces of nervous tissue first
in a solution of potassium dichromate (and usually osmic acid also) and then in silver nitrate.
Hematoxylin and Eosin (H&E): Hematoxylin is not a true basic dye. It is used with an
intermediary, which recognizes anionic tissue components. Hematoxylin is nearly a specific
stain for chromatin and it is therefore referred to as a "Basic" stain. It stains the nuclear
network, chromosomes, etc., blue. It is a regressive stain and is extracted by very dilute acid
or acid alcohol. It may be used after almost any fixative and is a permanent stain.
Eosin is a red general cytoplasmic stain. It combines with hemoglobin to give an orange color.
It is an acid dye and the terms acidophilic, oxyphilic and eosinophilic are often used
interchangeably. It may be used after any fixative and is used as a counter-stain in many
combinations in addition to hematoxylin.
Hematoxylin and Orange "G" (H & Or. G.): Orange G. is substituted for eosin. Acid orange-G
specifically stains the granules of acidophilic cells of the adenohypophysis.
Hematoxylin, Picric Acid and Acid Fuchsin (H & P.A.F.): See van Gieson.
Heidenhains Iron Hematoxylin: Chromatin material (nuclear network and chromosomes) blue
black. It is a popular cytological stain, especially for the study of mitosis. It can be used after
almost any fixative.
Injection (Inj.): The channels in the tissues, for example, blood and lymph vessels, are
injected with a colored mass. Berlin blue in dilute gelatin is a commonly used mass.
Mallory Azan: Collagenous fibers are blue. Nuclei are red. Cytoplasmic staining varies from
pink to reddish brown, depending upon the cell type.
Masson's Trichrome Stain (Masson's Tri.): Collagenous fibers blue. Muscle red. Nuclei red.
The sections are first stained with hematoxylin. They are then treated with ponceau red and
acid fuchsin, phosphomolybdic acid and aniline blue. You should be aware that in other
laboratories light green is used in place of aniline blue.
Nissl: A method of staining nucleic acids (e.g. ribosomes, RER, heterochromatin, nucleoli). A
dye such as methylene blue, toluidine blue or cresyl violet is used.
Orange "G" (Or. G.): A general cytoplasmic stain similar to eosin in action. Stains cytoplasm
yellow or orange.
Osmic Acid or Osmium Tetroxide (OsO4): A selective stain for unsaturated lipids and for
lipoproteins such as myelin, which it stains black. Also used as a fixative, especially for
electron microscopy.
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Periodic Acid Schiff and Hematoxylin (P.A.S. & H.): (See Junqueira 11th pp 12-13.)
Carbohydrates and carbohydrate compounds may be demonstrated by this histochemical
technique. Most carbohydrates react with periodic acid to produce aldehydes, which convert
the colorless Schiff reagent to pink, or magenta. Hematoxylin or methyl green is used to stain
the nuclei. Glycogen, mucin, elastic fibers, reticular fibers, basement membranes, thyroid
colloid, basophilic granules in the pituitary gland, and other polysaccharides such as the
ground substance of cartilage are stained fuchsia or pink.
Phloxin: This is a cytoplasmic stain belonging to the eosin series of dyes. It gives a reddish
tone to the cytoplasm.
Regaud's Hematoxylin for Mitochondria: Among the many methods used to demonstrate
mitochondria by light microscopy, the most permanent and the simplest is Regaud's
modification of iron hematoxylin on sections of material fixed in potassium dichromate and
formalin and subsequently mordanted in dichromate. After staining, the slides are
differentiated to remove the hematoxylin from most cytoplasmic components other than
mitochondria. Unfortunately, the results are not uniform: some cells will be over-stained and
some under-stained. Therefore a number of microscopic fields should be examined.
Silver nitrate (Ag.): The intercellular cement substance of epithelium is black. This is an
impregnation method. The fresh tissue is treated with silver nitrate and exposed to strong
light, which reduces the silver.
Silver Method for Golgi complex: Many methods have been used for staining the Golgi
complex of the cell. One of the best methods consists of direct fixation of fresh tissue in a
solution of silver nitrate in formalin, development in hydroquinone-formalin, followed by the
usual procedure for paraffin embedding and sectioning. In the slides prepared for the class
sets, the nuclei of the cells have been stained lightly by azocarmine.
Sudan Black: This is a stain that colors fat droplets black. There are several Sudan dyes,
among which are Sudan III and Sudan IV (Scarlet R.). These stain fat droplets red as does
Oil-red-O.
Supravital staining
A vital stain (e.g., trypan blue) is applied to an animal in life; a supravital stain (e.g., Janus
green, neutral red) is one that is applied to cells or tissues removed from the body. See the
first laboratory exercise, Introduction: Microscopy – Cytology for a description of the properties
of neutral red and Janus green vis a vis particular subcellular organelles.
Van Gieson: Collagenous fibers are yellow (e.g., dense connective tissue and bone), cartilage
matrix is brown.
Elastic Tissue Stain and Van Gieson's Picric Acid Fuchsin (Vieh. Van G. Verhoeff's): In
Verhoeff's stain the elastic tissue is black and in this combination with Van Gieson collagenous
tissue is red.
Weigert's Hematoxylin (W. Hem.): A modification of Heidenhain’s iron hematoxylin. Stains the
myelin sheath black.
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Weigert's Elastic Tissue Stain: Elastic fibers purple or black. It is a specially prepared
combination of basic fuchsin, resorcin, ferric chloride, water and alcohol.
Wright's Blood Stain (Wright): Nuclei blue or purple. Basophilic granules blue, acidophilic
granules red, and neutrophilic granules reddish lilac. Red blood corpuscles orange. The
eosinates of polychromed methylene blue are dissolved in absolute methyl alcohol. When this
solution is placed on a dried blood smear, the methyl alcohol acts as the fixative, and the
dissolved dye begins the staining process. After from one to three minutes, the stain is
diluted with an equal volume of distilled water. This differentially stains the cytoplasmic
granules and is allowed to act for about three minutes. It is then poured off and the
preparation is washed briefly in tap water and allowed to dry.
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THE MICROSCOPE
The following is intended to acquaint the student with the light microscope. The major parts of
the instrument will be named and a method for the effective use of the microscope will be
outlined. The comments apply to the Nikon student microscope but will apply directly, or with
slight modification, to student instruments of other manufacturers.
The microscope consists of a compound optical system (the objective lens and the ocular
lens); a movable specimen support (the mechanical stage); an illumination system (the lamp
and the condenser lens with its iris diaphragm). All the systems are attached to the
microscope stand consisting of the base and arm.
The microscopes used in the course have a binocular head, which may be rotated after
loosening its clamping screw. The topmost elements in the optical system are the eyepieces
or ocular lenses. The interpupillary distance may be varied. One of the eyepieces may have a
pointer, and note that one (or both) eyepiece(s) may be focused separately to compensate for
dioptric differences between your eyes.
The revolving nosepiece is the inclined, circular metal plate to which the objective lenses,
usually four, are attached. The objective lenses usually provide 4x, 10x, 40x and 100x
magnification. The final magnification is the product of the magnification of the ocular and
objective lenses. A slide is placed on the mechanical stage and is moved by rotating the stage
control knobs. The lamp is an integral part of the base. The fine and course focus controls
are mounted coaxially on the stand just above the base. The arm connects the base to the
binocular head-revolving nosepiece assembly.
The component of the illumination system immediately below the stage is the condenser. The
height of the condenser may be varied to give a bright, evenly illuminated field. Generally the
condenser is used in its highest position or just slightly lower. A lever projects from the
condenser and it is used to vary the opening of the condenser (or iris) diaphragm. For work
with the scanning (4x) and low-power (10x) objectives, the condenser diaphragm should be
wide open. For work with the high-dry (40x) and oil-immersion objectives (100x), however, the
diaphragm should be closed slowly while looking at a sharply focused section until the level of
illumination is just slightly reduced. This is the setting of the condenser diaphragm for
optimum contrast and resolution. (From a theoretical point of view this is not quite correct. The
diaphragm should be adjusted for each magnification. In most instances, however, it is much
less critical at the lower magnifications).
In examining a slide with the light microscope, the following sequence of steps should be
used:
1) Place the slide on the stage and examine it with the scanning objective (4x). Scan the
entire section. Often tissue and organ identification can be made at this magnification.
Select an area or areas for study at higher magnification.
2) Rotate the revolving nosepiece to place the lower-power objective (10x) in the optical
axis. When turning the nosepiece, grasp the nosepiece itself or the part of the
objective adjacent to the nosepiece to avoid excess stress on the objective.
3) Proceed to the next step in magnification, if necessary, which is high dry (40x). Adjust
the condenser.
4) For some specimens, especially blood and cellular organelles, you may want to use
the highest magnification, which is oil immersion (100x). The following procedure must
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be used when working with the oil immersion lens: a) focus carefully on a selected area
with the high-dry objective, b) swing the high-dry objective out of the light path and
allow the nosepiece to remain in an intermediate position between the high-dry and the
oil-immersion objectives, c) place a drop of immersion oil (available in the bookstore)
on the slide in the appropriate region to be studied, d) swing the oil-immersion
objective into position. The distance between the front element of the objective and the
surface of the slide will be about 1.0mm, and the oil will form a bridge between the
slide and the objective. The area to be examined should be within the field and should
require only slight refocusing. You may need to readjust the condenser.
When finished using the oil-immersion objective, both the objective and the slide must be
wiped with lens paper (available in the bookstore). If oil is allowed to dry on the high-dry or oil-
immersion objective, the optical performance of the instrument will be severely reduced. The
dried oil must be removed with lens paper.
Be careful not to move the high-dry objective into position while oil is on the slide. If this is
done by mistake, the high-dry objective must be cleaned by wiping the front element with lens
paper.
Slides should be cleaned with lens paper or tissue to remove fingerprints, oil or dirt. If the slide
cannot be cleaned with a dry tissue, use alcohol.
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